INVESTIGATING THE ROLE OF TYROSINE KINASE RECEPTORS TIE2 AND
VEGFR2 IN CANCER
A thesis
Presented to
The Faculty of Graduate Studies
of
The University, of Guelph
by
UNA ADAMCIC
In partial fulfillment of requirements
for the degree of
Doctor of Philosophy
May, 2010
©Una Adamcic, 2010 Library and Archives Biblioth6que et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'6dition
395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada
Your file Votre r6f6rence ISBN: 978-0-494-67853-4 Our file Notre reference ISBN: 978-0-494-67853-4
NOTICE: AVIS:
The author has granted a non- L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliothdque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Nnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, glectronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats.
The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre im primes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission.
In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these.
While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis.
••I Canada ABSTRACT
INVESTIGATING THE ROLE OF TYROSINE KINASE RECEPTORS TIE2 AND VEGFR2 IN CANCER
Una Adamcic Advisor: University of Guelph, 2010 Dr Brenda Coomber
To better understand tumor-related angiogenesis, we studied two critical tyrosine kinase receptors expressed on endothelial cells, Tie2 and VEGFR2. These play important roles in formation and stability of blood vessels during the process of angiogenesis.
Targeted and non-targeted antiangiogenic therapies were employed to inhibit these pathways, in order to determine their function in tumor vasculature. When administered alone, these therapies showed only transient anti-tumor properties, but when combined, these therapies "cancel" each other, suggesting that the interaction of these pathways is complex and needs to be studied in more detail. We also identified the presence of
VEGFR2 expression in the membrane and nucleus of melanoma cell lines and determined their contribution to both autocrine and intracrine VEGF/VEGFR2 signaling.
Besides signaling survival pathways, we suggest the role of melanoma cell VEGFR2 to be primarily to aid in migration and adhesion, seen through the decreased phosphorylation of focal adhesion kinase when cells were exposed to small-molecule inhibitor ZM323881. In fact, ZM323881 was a more potent inhibitor of VEGFR2 signaling than bevacizumab, an anti-VEGF neutralizing antibody. Although bevacizumab showed sustained increase in phosphorylation of VEGFR2 in vitro, when tested in vivo, it had an antiangiogenic effect in melanoma xenografted tumor.
Finally, we identified bone-marrow derived circulating endothelial cells in bovine blood that differentiate in vitro into endothelial cells that display differential expression of Tie2 and VEGFR2, and are able to home to Matrigel plugs when injected into immune deficient mice. These studies give us a better understanding not only about the expression of Tie2 and VEGFR2 receptors in endothelial cells, but also in circulating endothelial cells from peripheral blood and cancer cells themselves. ACKNOWLEDGEMENTS
First and foremost, I would like to thank my supervisor and mentor, Dr Brenda
Coomber for all her encouragement and her guidance in these past six years. Thank you for being so supportive of me and my scientific ideas as well as providing support and understanding during some difficult times in my life. I am also grateful to the members on my committee, Dr. Roger Moorehead, Dr. Jim Petrik and Dr. Paul Woods, for advice on preparation of this thesis as well as your kind words during these past years of my stay in the department.
I would also like to thank the staff and the students of the Department of
Biomedical Sciences and Central Animal Facility, for all your help and support.
Thanks to all my project students - Valerie, Alex, Craig, Freda, Clorinda,
Whitney and Hassan, who helped me on various projects throughout these years.
Special thanks to the whole Coomber Lab, past - Courtney Stone, Dr Claire
Plumb, Lindsay Quayle, Kristen Lacombe, Steve Patten, Mackenzie Smith, Andy
Bertolini ("adopted" lab member from Dr Ann Hahnel's lab), Dr. Sira Shahrzad, Dr. Sirin
Adham Yeeish, and Dr. Ifat Sher - and present - Kaya Skowronski, Lizzie Kuczynski,
Jen Thompson and Kanwal Minhas. All these people were my family for the past six years and their friendships had a strong impact on my life. We've been together though just about everything - babies, weddings, break-ups and new loves, Relays for Life, lots of parties and many, many, more wonderful things.
Last but not least I would like to thank my family - my mom Ana, my dad Aloys and my "skinny little thing", cat Martin, as well as my soon-to-be husband Sanin and our two "fluff balls", cats Phillipe and Timmy. I would never be where I am without your
i unconditional love and support throughout these years. You guys were behind me every step of the way and this thesis is dedicated to you and your hard work - for putting up with me all these years. I love you all.
ii DECLARATION OF WORK PERFORMED
I declare that with the exception of the items indicated below, all work reported in this thesis was performed by me.
Under my supervision, Clorinda Castagna performed sectioning, staining and quantification of necrotic and hypoxic areas of WM239 xenografts treated with
TekdeltaFc and low dose metronomic cyclophosphamide. Kanwal Minhas performed immunofluorescent staining of the same tumors using CD31 /desmin/PDGFRP antibodies.
Primers for VEGFR2 mRNA used for PCR analysis of melanoma cell lines were designed by Dr Ifat Sher. Preliminary western blots for basal expression of VEGFR2 in melanoma cell lines compared to BAECs was performed by Craig Peters. Freda Tam performed studies on Thrombospondin-1 expression in conditioned media of melanoma cell lines grown in normoxic or hypoxic conditions, with and without 4- hydroxycyclophosphamide, and performed all western blots regarding TSP-1. Alex
Yurkievich performed preliminary studies on bovine endothelial progenitor cell isolation.
Dr Brenda Coomber did all subcutaneous cell injections into mice used for xenograft studies. She also injected TekdeltaFc twice a week subcutaneously. Jackie
Rombeek did tail vein injections of bovine endothelial clones into mice bearing Matrigel plugs, as well as bevacizumab treatments twice a week. Karolina Skowronski and
Mackenzie Smith helped generously in the collection of tumor xenografts from all mouse trials.
iii TABLE OF CONTENTS ACKNOWLEDGEMENTS I DECLARATION OF WORK PERFORMED Ill LIST OF FIGURES VII LIST OF ABBREVIATIONS IX INTRODUCTION 1 CHAPTER 1 - LITERATURE REVIEW 4 VASCULAR DEVELOPMENT 5 Vasculogenesis 5 Angiogenesis 5 Postnatal Vasculogenesis 7 ANGIOGENESIS AND CANCER 9 The Angiogenic Switch 10 Structure and Function of Tumor Vasculature 11 CANCER TREATMENT THERAPIES 12 Maximum Tolerated Dose vs. Metronomic Low-Dose Chemotherapy 12 Antiangiogenic Therapy 14 Angiogenic Inhibitors in Combination with Chemotherapeutic Agents 16 TIE2, ENDOTHELIAL TYROSINE KINASE RECEPTOR 16 Tie2 and Angiopoietins - Function and Structure 17 Lessons Learned From Knockout and Overexpression Studies 19 Significance of Tie! signaling transduction pathway 20 Role of Tie2 in Cancer 22 VEGF PATHWAY IN TUMOR ANGIOGENESIS 24 Vascular Endothelial Growth Factor 24 Signaling through Vascular Endothelial Growth Factor Receptors 26 Recent Developments in VEGFR2 Targeted Therapies 28 THE ROLE OF THROMBOSPONDIN-1 IN CANCER 30 AUTOCRINE AND INTRACRINE SIGNALING OF RECEPTOR TYROSINE KINASES 32 RATIONALE 35 CHAPTER 2 - FUNCTIONAL AUTOCRINE AND INTRACRINE VEGF/VEGFR2 SIGNALING LOOP IN MALIGNANT MELANOMA 37 INTRODUCTION 38 MATERIAL AND METHODS 41 Cell lines and culture conditions 41 Establishment of tumor xenografts 41 Reverse transcription/polymerase chain reaction (RT-PCR) 42 Western blotting 43 Measurement of human VEGF protein levels in melanoma cell conditioned medium 45
VI Immunofluorescence for phosphorylated and native VEGFR2 receptor in cultured cells 45 VEGFR2 immunostaining & blood vessel density quantification 46 Statistical Analysis 47 RESULTS 48 VEGF and VEGFR2 expression in primary and metastatic melanoma cell lines in vitro 48 Neutralization of VEGF-A ligand in vitro 48 Functional autocrine and intracrine VEGF/VEGFR2 signaling loops 49 Bevacizumab is antiangiogenic in primary and metastatic melanoma xenografts ...51 Downregulation of VEGFR2 in severe hypoxia 52 DISCUSSION 53 CHAPTER 3 - ANTIANGIOGENIC ACTIVITY OF METRONOMIC CYCLOPHOSPHAMIDE IN HUMAN MELANOMA XENOGRAFTS IS MODULATED BY TIE2 INHIBITION 74 INTRODUCTION 75 MATERIAL AND METHODS 80 Cell lines and reagents 80 Treatment using TekdeltaFc and 4HC in vitro 80 Exposure of endothelial cells to hypoxic, hypoglycemic and cancer cell conditioned medium 81 Western blotting 82 Growth of tumor xenografts 83 Quantification of tumor hypoxic and necrotic areas 84 Quantification of Tie 2 expression and microvessel density 86 Quantification of blood vessel pericyte coverage 87 Measurement of VEGF levels in tumor xenograft lysates 88 Measurement of Thrombospondin-1 levels in conditioned media 88 Statistical Analysis 88 RESULTS 90 The effects ofTie2 inhibitor and 4HC on survival and cord formation on Matrigel of endothelial cells in vitro 90 Effects of LDM CTXand Tie2 inhibitor in xenograft model 90 Tie2 inhibitor significantly increased pericyte coverage of tumor blood vessels compared to low dose CTX treatment 91 TekdeltaFc and LDM CTX increase VEGF expression 92 The effects of Tie2 inhibitor and 4HC on Tie2 signaling transduction in vitro 92 Hypoxia induces downregulation of Tie2 expression in vitro 92 TSP-1 levels are modulated by LDM CTX and hypoxia 93 DISCUSSION 94 CHAPTER 4 - DIFFERENTIAL EXPRESSION OF TIE2 AND VEGFR2 RECEPTORS IN CLONES DERIVED FROM ISOLATED BOVINE ENDOTHELIAL PROGENITOR CELLS 114 INTRODUCTION 115 MATERIAL AND METHODS 118
v Buffy-Coat Preparation 118 Culture of MNCs to obtain EPC derived colonies 119 Western Blot Analysis 120 Fluorescent Labeling Using Cell Tracker Probes 121 In vivo Matrigel Plug Assay 121 Visualization of injected cells 122 Statistical analysis 122 RESULTS 123 Phenotypic differences in cells arising from bovine peripheral blood MNCs 123 Characterization of expanded colonies reveals differential expression of receptor tyrosine kinases 124 Clones with differential expression of Tie2/VEGFR2 home to sites of angiogenesis in Matrigel plug assay in vivo 125 DISCUSSION 126 GENERAL DISCUSSION 137 SUMMARY AND CONCLUSIONS 147 REFERENCES 149 APPENDIX I - CHEMICAL LIST AND SUPPLIERS 182 APPENDIX II - PREPARATION OF MATERIALS USED 185
VI LIST OF FIGURES FIGURE 1. EXPRESSION OF VEGFR2 IN PRIMARY AND METASTATIC MELANOMA CELLS IN VITRO 61 FIGURE 2. EFFECTS OF BEVACIZUMAB ON THE VEGF EXPRESSION OF PRIMARY AND METASTATIC MELANOMA W VITRO 62 FIGURE 3. DETECTION OF PHOSPHORYLATED VEGFR2 RECEPTOR IN PRIMARY AND METASTATIC MELANOMA LYSATES 63 FIGURE 4. DETECTION OF PHOSPHORYLATED VEGFR2 RECEPTOR IN PRIMARY AND METASTATIC CULTURED MELANOMA CELLS 64 FIGURE 5. INHIBITION OF VEGFR2 PHOSPHORYLATION IN PRIMARY MELANOMA CELL CYTOSOLIC FRACTIONS IN VITRO 65 FIGURE 6. DETERMINATION OF THE INHIBITION OF VEGFR2 PHOSPHORYLATION IN PRIMARY MELANOMA CELLS NUCLEAR FRACTIONS IN VITRO 66 FIGURE 7. LONG-TERM INHIBITION OF VEGFR2 PHOSPHORYLATION IN PRIMARY MELANOMA CELLS IN VITRO 67 FIGURE 8. INHIBITION OF VEGFR2 PHOSPHORYLATION IN METASTATIC MELANOMA CELLS IN VITRO 68 FIGURE 9. INHIBITION OF VEGFR2 PHOSPHORYLATION IN METASTATIC MELANOMA CELLS NUCLEAR FRACTIONS IN VITRO 69 FIGURE 10. IMPACT OF BEVACIZUMAB ON GROWTH OF PRIMARY AND METASTATIC MELANOMA CELL XENOGRAFTS 70 FIGURE 11. IMPACT OF BEVACIZUMAB ON ANGIOGENESIS OF PRIMARY AND METASTATIC MELANOMA CELL XENOGRAFTS 71 FIGURE 12. EXPRESSION OF VEGFR2 IN MELANOMA XENOGRAFTS TREATED WITH AND WITHOUT BEVACIZUMAB 72 FIGURE 13. EXPRESSION OF VEGFR2 RECEPTOR IN PRIMARY AND METASTATIC MELANOMA CELL LINES EXPOSED TO HYPOXIC CONDITIONS IN VITRO 73 FIGURE 14. ENDOTHELIAL CELL SURVIVAL AFTER TREATMENT WITH TEKDELTAFC AND 4- HYDROXYCYCLOPHOSPHAMIDE (4-HC) IN VITRO 100 FIGURE 15. EFFECTS OF TEKDELTAFC ON FORMED ENDOTHELIAL CELL CORDS ON MATRIGEL 101 FIGURE 16. ENDOTHELIAL CELL CORD FORMATION ON MATRIGEL IN THE PRESENCE OF TEKDELTAFC IN VITRO 102 FIGURE 17. IN VIVO XENOGRAFTS OF HUMAN MELANOMA CANCER TREATED WITH TEKDELTAFC AND LOW DOSE METRONOMIC CYCLOPHOSPHAMIDE 103 FIGURE 18. QUANTIFICATION OF HYPOXIC AND NECROTIC AREAS IN TUMOR XENOGRAFTS TREATED WITH TEKDELTAFC AND LDM CTX 104 FIGURE 19. DUAL IMMUNOFLUORESCENT STAINING OF MELANOMA XENOGRAFTS FOR CD31 AND TIE2 105 FIGURE 20. QUANTIFYING % TIE2 NEGATIVE BLOOD VESSELS AND BLOOD VESSEL DENSITY IN TUMOR XENOGRAFTS TREATED WITH TEKDELTAFC AND LDM CTX 106 FIGURE 21. IMMUNOSTAINING OF BLOOD VESSELS AND PERICYTES IN TREATED TUMORS. 107 FIGURE 22. QUANTIFYING THE AMOUNT OF BLOOD VESSEL PERICYTE COVERAGE IN TEKDELTAFC AND LDM CTX TREATED TUMORS 108 FIGURE 23. EXPRESSION OF VEGF (PG/ML/MG) IN TUMOR XENOGRAFT LYSATES 109
vii FIGURE 24. CHANGE IN NATIVE AND PHOSPHORYLATED TIE2 IN BAEC TREATED WITH TEKDELTAFC AND 4-HC IN VITRO 110 FIGURE 25. EXPRESSION OF TIE2 RECEPTOR IN HYPOXIA AND HYPOGLYCEMIA IN VITRO ..111 FIGURE 26. EXPRESSION OF TIE2 RECEPTOR BY VESSELS IN HYPOXIC AND NORMOXIC AREAS OF TUMOR XENOGRAFTS 112 FIGURE 27. EXPRESSION OF TSP-1 IN CONDITIONED MEDIUM FROM HUMAN PRIMARY AND METASTATIC MELANOMA CELL LINES 113 FIGURE 28. PHENOTYPE OF CELLS ARISING FROM PERIPHERAL BLOOD MNCS 130 FIGURE 29. WESTERN BLOT ANALYSIS OF ISOLATED BOVINE CLONES GROW ON DIFFERENT EXTRACELLULAR MATRICES 131 FIGURE 30. PHENOTYPE OF CLONES WITH A DIFFERENTIAL EXPRESSION OF TIE2/VEGFR2 RECEPTORS 132 FIGURE 31. EXPRESSION OF CD45 MARKER BY ISOLATED ENDOTHELIAL CLONES 133 FIGURE 32. ABILITY OF CLONES WITH DIFFERENT EXPRESSION OF TIE2 AND VEGFR2 RECEPTORS TO FORM CORD-LIKE STRUCTURES ON MATRIGEL 134 FIGURE 33. DETECTION OF FLUORESCENTLY LABELED CLONES 135 FIGURE 34. INFILTRATION OF ENDOTHELIAL CELLS AND BLOOD VESSEL FORMATION IN MATRIGEL PLUG 136
Vlll LIST OF ABBREVIATIONS
Abbreviation Full name Ang Angiopoietin 4-HC 4-hydroxycyc lopho sphamide ATCC American Type Culture Collection BAEC Bovine Aortic Endothelial Cells BSA Bovine Serum Albumin BVD Blood Vessel Density C Control CM Conditioned Media CTX Cyclophosphamide DAPI 4', 6-diamidino-2-phenylindole DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic acid EC Endothelial Cell ECM Extracellular Matrix EGF Endothelial Growth Factor EGFR Endothelial Growth Factor Receptor EPC Endothelial Progenitor Cell FAK Focal Adhesion Kinase FBS Fetal Bovine Serum FDA Food and Drug Administration FdG PET 18-fluorodeoxyglucose positron-emission tomography FGF Fibroblast Growth Factor FGFR Fibroblast Growth Factor Receptor FU Fluorouracil G-CSF Granulocyte Colony Stimulating Factor GFP Green Fluorescent Protein HIF Hypoxia Inducible Factor HRE Hypoxia Response Element IL Interleukin LDM CTX Low dose metronomic cyclophosphamide LG Low Glucose LO Low oxygen LOLG Low oxygen low glucose MMP Matrix Metalloproteinase MTD Maximum Tolerated Dose NO Nitric Oxide NSCLC Non-small Cell Lung Carcinoma PBS Phosphate Buffer Saline PCR Polymerase Chain Reaction
IX Abbreviation (Continued) Full name (Continued) PDGF Platelet-derived Growth Factor PDGFR Platelet-derived Growth Factor Receptor PFA Paraformaldehyde PMSF Phenylmethanesulfonyl fluoride POD Peroxydase PVDF Polyvinyl difluoride RT Room temperature RTK Receptor Tyrosine Kinase SDF Stroma derived factor SDS-PAGE Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis sTie2 Soluble Tie2 TBS Tris-buffered Saline TGF Transforming Growth Factor TNF Tumor necrosis factor TSP-1 Thrombospondin-1 VEGF Vascular Endothelial Growth Factor VEGFR Vascular Endothelial Growth Factor Receptor
X INTRODUCTION
According to the Canadian Cancer Society an estimated 171,000 new cases of cancer and 75,300 deaths from cancer occurred in Canada in 2009 (www.cancer.ca). At least 55% of those new cases are due to prostate, lung and colorectal cancers in men, and breast, lung and colorectal cancers in women. These statistics are not reflective of Canada alone - worldwide, cancer is a leading cause of death for both men and women, in particular lung and colorectal cancers. On the basis of current mortality rates, 24% of women and 29% of men, or approximately 1 out of every 4 Canadians, will die from cancer. Even with considerable improvements in patient survival rates attributed to the improvement of chemotherapeutic agents over the years, the problems with acquired and intrinsic resistance in solid tumors still exist, suggesting a need for novel approaches in treating cancer.
One of these novel approaches emerged more than three decades ago when angiogenesis, or the growth of new blood vessels, was recognized as a target in cancer treatment based on the fact that without angiogenesis, tumors cannot grow and metastasize (Folkman, 1971). The first antiangiogenic therapy was approved by the USA
FDA in 2004 when bevacizumab (AvastinR) (www.fda.gov), a monoclonal antibody targeting vascular endothelial growth factor (VEGF), prolonged survival of patients with advanced cancer in a large clinical trial (Cohen et al., 2007; Ferrara et al., 2005; Garcia-
Saenz et al., 2008; Hurwitz et al., 2004; Willett et al., 2004; Yang et al., 2003). Despite the encouraging signs from early preclinical studies, most antiangiogenic therapies failed to generate the same effectiveness in clinical trials, perhaps due to an incomplete understanding of the angiogenic processes and the role of the tumor microenvironment.
1 Thus, there is a growing interest in understanding these complex mechanisms of
antiangiogenic drug resistance. In many cases it is unclear if a patient's cancer expresses the target of the antiangiogenic drug or the extent of interaction between this target and
the antiangiogenic drug administered. The complexity of tumor vasculature is
additionally complicated by the contribution of bone marrow derived haematopoietic and
endothelial progenitor cells as well as cells from other organ specific vascular niches (De
Palma and Naldini, 2006; Ito et al., 1999; Nolan et al, 2007; Walter and Dimmeler,
2002). Due to this complexity, therapies currently used in clinical trials are composed of
various combinations of standard and metronomic dose schedules with a multitude of
antiangiogenic agents, targeting various pathways utilized in tumor development.
Interactions between tumor and endothelial cells influence tumor vessel
phenotype and its behavior. Expression of endothelial tyrosine kinase receptor, Tie2, was
found to be heterogeneous and tumor-type specific (Fathers et al., 2005). Moreover, Tie2
negative blood vessels were found to increase with tumor metastatic progression and
influence tumor response to targeted and non-targeted antiangiogenic therapies (Fathers
et al., 2005). Therefore, studying the Tie2 signaling transduction pathway in tumor
vasculature may give us an important insight into not only the complexity of tumor-
endothelial interactions and how they differ between different tumor types, but also some
answers on how to improve antiangiogenic therapy outcomes.
In addition, the equally important VEGF/VEGFR2 signaling pathway has
received a huge amount of attention in the last decade and the majority of antiangiogenic
drugs that are in clinical trials today, target certain components of this pathway. In fact,
some of them, such as small molecule multi receptor tyrosine kinase inhibitors, target
2 many tyrosine kinase downstream signaling pathways simultaneously. Nonetheless, these therapies also produced only modest outcomes for cancer patients suggesting that these angiogenic molecule pathways should be studied in more depth in cancer (Backer et al.,
2009; Ferrara et al., 2005; Hsu and Wakelee, 2009; Ivy et al., 2009; Pytel et al., 2009;
Quesada et al., 2007; Smith et al, 2004; Steeghs et al., 2007; Takimoto, 2009). Since many of the cancers were shown to produce VEGF ligand and VEGF receptors, there is a need to determine the mechanism and the function of these pathways on these cells.
Therefore, receptor tyrosine kinases play an important role in sustaining the growth and progression of most cancer types and should be studied in more depth in this pathological conditions in order get a better understanding of their functioning that may eventually lead to better cancer therapies.
3 CHAPTER 1 - Literature Review
4 VASCULAR DEVELOPMENT
Vasculogenesis
Blood vessels are constructed by two processes - vasculogenesis and angiogenesis. Vasculogenesis occurs in the developing embryo, where first blood vessels are created de novo from lateral plate mesoderm (Risau, 1997; Wilting and Christ, 1996).
Hemangioblasts form aggregations called blood islands, where cells at the periphery, angioblasts, give rise to vascular endothelial cells (ECs), while cells in the center of the blood island, haematopoietic stem cells, give rise to mature blood cells. Three growth factors are responsible for initiating vasculogenesis - basic fibroblast growth factor
(Ribatti et al., 1995), required for generation of hemangioblasts from mesoderm (Flamme and Risau, 1992); vascular endothelial growth factor, that enables the differentiation of angioblasts and their formation into endothelial cells (Fong et al., 1995; Millauer et al.,
1993; Risau and Flamme, 1995; Shalaby et al., 1995) and angiopoietin-1, that mediates interaction of endothelial cells and pericytes (Hayes et al., 1999; Suri et al., 1996). Fusion of blood islands in the embryo initiates the formation of the capillary network structure, called the primary capillary plexus. This formed capillary plexus is remodeled by sprouting and branching of new vessels from pre-existing ones, a process known as angiogenesis (Korpelainen and Alitalo, 1998; Munoz-Chapuli et al., 2004; Risau, 1997).
Angiogenesis
In an adult, angiogenesis is initiated by production of angiogenic stimuli (such as
VEGF) by host cells that require blood vessel recruitment (Carmeliet and Jain, 2000). An
5 increase in VEGF production causes an increase in vascular permeability (Bates and
Harper, 2002; Brown et al., 1992; Zeng et al, 2001a). To stimulate ECs migration, mature vessels secrete Angiopoietin-2 (Ang-2), a Tie2 receptor tyrosine kinase antagonist that causes detachment of smooth muscle cells (in larger vessels) and pericytes (in capillaries), resulting in a temporary blood vessel destabilization (Maisonpierre et al.,
1997). Extracellular matrix is degraded by matrix metalloproteineses (MMPs), allowing the "tip" endothelial cells to start migrating through degraded matrix where they are followed by proliferating endothelial "stalk" cells. The family of VEGFs and their receptors are major regulators of sprouting angiogenesis (Gerhardt et al, 2003; Ruhrberg et al, 2002). Finally, through cell-cell contact and cell-ECM interactions, capillary loops form and endothelial cells become polarized, forming a lumen (Hanahan and Folkman,
1996).
These mature and functional blood vessels have to be stabilized by perivascular cells (pericytes and smooth muscle cells). This 'cross-talk' between ECs and pericytes is critical for proper vessel development and maturation. Binding of platelet-derived growth factor-(3 (PDGF-B), secreted by both endothelial and non-endothelial cells, to its receptor
PDGFR-P, causes pericyte recruitment to the sprouting EC tubes (Abramsson et al.,
2003). It also causes an increase in Angiopoietin-1 (Ang-1), a Tie2 agonist, which when bound to Tie2 establishes pericyte-EC interaction (Betsholtz, 2004; Hellstrom et al.,
1999; Lindblom et al., 2003). Finally, once pericytes and ECs are in contact, PDGF-P stimulates pericytes to secrete transforming growth factor-(3 (TGF-P) that in turn inhibits further EC proliferation (Nishishita and Lin, 2004).
6 Once assembled into new vessels, EC become quiescent and survive for years
(Peters et al., 2004; Wong et al., 1997). Due to this, ECs are among the most quiescent cells in the body outside the central nervous system, with only 1 in every 10,000 (0.01%)
ECs, in comparison with about 14% of normal intestinal epithelial cells, in the cell division cycle at any given time (Hobson and Denekamp, 1984). This EC division is tightly regulated by the fine balance between endogenous angiogenic activators and inhibitors, a mechanism known as the "angiogenic switch" which, when turned on, signals normally quiescent vasculature to sprout new capillaries (Hanahan and Folkman,
1996). In adults, angiogenesis occurs under a limited number of physiological conditions such as ovulation, menstruation, pregnancy and wound healing (Brown et al., 1992;
Carmeliet and Jain, 2000). Unfortunately, it also occurs in some pathological conditions, for instance in diabetic retinopathy (Caldwell et al., 2003; Takagi et al., 2003), psoriasis
(Brown et al., 1995; Detmar et al., 1994), arthritis (Gravallese et al., 2003) and tumor growth (Folkman, 1971; Ruiter et al., 1993).
Postnatal Vasculogenesis
In contrast to the original thinking that adult neovascularization is primarily achieved through the process of angiogenesis, studies have identified a population of bone marrow-derived endothelial progenitor cells (EPCs) that enter the peripheral blood and insert themselves at the sites of active angiogenesis. This unique process is now referred to as postnatal vasculogenesis. The participation of EPCs in tumor neovascularization has been reported in numerous studies (Asahara et al., 1999; Davidoff et al., 2001; Shi et al., 1998; Takahashi et al., 1999; Patenaude et al, 2010). The first
7 report was that of Asahara and colleagues (Asahara et al., 1997), where murine colon cancer cells were injected into mosaic mice with bone marrow transplanted from animals that had constitutive expression of P-galactosidase gene regulated by an endothelial cell- specific promoter. The histological examination of the tumors revealed multiple LacZ+ cells within tumor stroma and incorporated into the endothelial layer of tumor blood vessels. Since then, several recent studies evaluated the role of bone marrow derived endothelial cells in establishing a niche for metastatic tumor cell engraftment and their role in progression of small to large tumor masses (De Palma and Naldini, 2006; Kaplan et al, 2005; Nolan et al., 2007; Walter and Dimmeler, 2002).
Despite many controversial studies, most researchers consider EPCs to be functionally and phenotypically distinct from mature ECs, with an ability to differentiate into mature ECs with all expressional and functional characteristics in vitro, and contribute to vasculogenesis and vascular homeostasis in vivo. EPCs have been implicated in processes such as repair of myocardial ischemia and infarction (Kalka et al.,
2000b; Orlic et al., 2001; Shintani et al., 2001), revascularization during limb ischemia
(Takahashi et al., 1999), and tumor vascularization. It is speculated that signals for EPC mobilization come from the periphery and are initiated by ischemia or vessel wall damage after which an increased number of circulating EPCs can be observed in the blood of the individual (Takahashi et al., 1999; Real et al, 2008). Their therapeutic potential is limited by EPC number - healthy adult humans are reported to have 2.6 ±1.6 circulating EPCs per mL of peripheral blood (Solovey et al., 1997). Studies in both mice and humans reveals that these numbers may be increased by administration of granulocyte-colony stimulating factor (G-CSF) (Natori et al., 2002; Yoshioka et al.,
8 2006), VEGF (Kalka et al., 2000b), estrogen (Iwakura et al., 2003) and stroma-derived factor-1 (SDF-1) (Naiyer et al., 1999). The number of EPCs that participate in the
formation of new blood vessels is still not clear (Walter and Dimmeler, 2002), with reports ranging from very low (<0.1%)(Larrivee et al., 2005; Purhonen et al., 2008) to high (up to 50%)(Nolan et al., 2007) percentage of EPCs that incorporate into newly
formed blood vessels, most likely dependent on the type of angiogenesis model used.
ANGIOGENESIS AND CANCER
In humans, cancer formation is a multi-step process reflecting genetic alterations leading to progressive transformation of normal human cells into pre-malignant states to
finally highly malignant invasive cancer cells (Hanahan and Weinberg, 2000). In fact,
Hanahan and Weinberg suggested that most of the cancer cell phenotypes are a manifestation of six essential alterations in cell physiology: (1) self-sufficiency in growth signals, (2) insensitivity to growth inhibitory signals, (3) evasion of programmed cell
death (apoptosis), (4) limitless replicative potential, (5) tissue invasion and metastasis and
(6) sustained angiogenesis.
Generation of tumor blood supply is a rate-limiting step in tumor progression and one of the main hallmarks of cancer (Hanahan and Weinberg, 2000). Initially, before attaining a blood supply, most primary solid tumors go through a prolonged state of avascular and possibly dormant growth, reaching up to 1-2 mm in diameter, attaining nutrients and oxygen through simple passive diffusion (Folkman, 1971; Ruiter et al.,
1993). Despite numerous findings that most tumors arise in this avascular manner, recent
9 studies suggest that many tumors, in particular secondary metastases do not initiate in this way. The tumors can also grow by host blood vessel co-option, thus starting off as well- vascularized small tumors (Holash et al., 1999). In response, host vessels secrete high amounts of Ang2, making these vessels regress. This results in cutting off of tumor vascular supply, creating a tumor that is avascular and hypoxic. Unfortunately, many tumors manage to overcome this host vessel regression by inducing angiogenesis primarily by secretion of VEGF.
The Angiogenic Switch
Normal and pathologic angiogenesis is tightly regulated by pro- and antiangiogenic growth factor signals. Proangiogenic factors, such as VEGF, fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF) stimulate angiogenesis and are produced by tumor or inflammatory cells into the tumor microenvironment (Tsai et al., 1995). Turning an 'angiogenic' switch 'on' results in a stimulation of surrounding mature host blood vessels to begin sprouting new blood vessel capillaries towards the tumor, eventually infiltrating it (Bergers and Benjamin, 2003; Hanahan and Folkman,
1996). It is now widely accepted that the 'angiogenic switch' is 'off' when the effect of pro-angiogenic molecules is balanced by that of antiangiogenic molecules, and is 'on' when the net balance is tipped in favor of angiogenesis (Hanahan and Weinberg, 2000).
To date, various signals have been identified that trigger the angiogenic switch including secreted proteins (epidermal growth factor, insulin-like growth factor, interleukins)
(Bunone et al., 1999), metabolic stress (hypoxia or low pC>2, acidosis or low pH, and glucose deprivation) (Shweiki et al, 1995; Tsai et al., 1995), mechanical stress,
10 immune/inflammatory response (cyclooxygenase-2, prostaglandins) (Coussens et al,
1999) and genetic mutations causing activation of oncogenes, such as K-ras and H-ras, c- myc and N-myc, b-raf, HER-2/neu, c-JUN (Denhardt, 1996; Grugel et al., 1995;
Kerkhoff and Rapp, 1998; Lehman et al., 1991; Rak et al., 1995; Rapp et al., 1986;
Suarez, 1989; Zou et al., 1997) and/or deletion of tumor-suppressor genes, such as p53
(Dameron et al., 1994) and pl6 (Liggett and Sidransky, 1998). These pro- and antiangiogenic molecules may be produced by cancer cells, ECs or stromal cells. These molecules represent attractive targets currently investigated in treating cancer
(Ramanujan et al., 2000).
Structure and Function of Tumor Vasculature
The abnormal features of tumor vasculature vary from tumor to tumor, and are probably due to the disproportionate expression of angiogenic growth factors and
inhibitors (Bergers and Benjamin, 2003). Tumor blood vessels are highly disorganized, tortuous, dilated and excessively branched with numerous shunts. Due to their uneven diameter, their blood flow is chaotic and variable (Baish and Jain, 2000), leading to the establishment of hypoxic and acidic regions within the tumor. This leads to the selection of highly malignant and metastatic cells that lose their apoptotic response to hypoxia
(Giaccia et al., 1998). Tumor blood vessel walls are also known to be leaky by having numerous "openings" (endothelial fenestrae, vesicles and transcellular holes), wide inter- endothelial junctions and discontinuous or absent basement membranes (Hashizume et al., 2000). The ECs that form these walls are abnormal in shape, grow on top of each other and project into the lumen.
11 In addition, pericytes on tumor vessels have multiple abnormalities (Morikawa et al., 2002). Unlike the tight association of pericytes and ECs in normal vessels, pericytes are only loosely associated with the endothelium of tumor vessels (Morikawa et al.,
2002). Moreover, they have abnormal shapes and may extend their cytoplasmic processes away from the vessel wall. These pericyte abnormalities in tumors are likely due to the alterations in PDGF signaling pathways (Abramsson et al., 2003). Mice deficient in
PDGF-B or its receptors have blood vessels with loose pericyte attachment, irregular diameter, luminal projections of endothelial cells and hemorrhage, similarly as observed in tumor vessels (Betsholtz, 2004; Hellstrom et al., 1999; Lindblom et al., 2003).
CANCER TREATMENT THERAPIES
Maximum Tolerated Dose vs. Metronomic Low-Dose Chemotherapy
Cytotoxic chemotherapeutic drugs cause DNA damage and disrupt DNA replication in proliferating cells and are often used at maximum tolerated doses to kill rapidly dividing tumor cells without life-threatening levels of toxicity (Takimoto, 2009). They are usually administered with prolonged (2-3 week) breaks between therapy cycles due to numerous side effects including acute myelosuppression, hair loss, damage to intestinal mucosa, nausea and mucositis, as well as long-term cardiac, renal, neurological and reproductive consequences (Gutierrez et al., 2009). Despite numerous clinical trials involving a multitude of such agents, tumor growth suppression induced by such agents is usually short-lived with relapses marked by development of very aggressive cancers due to their resistance to these cytotoxic drugs (Takimoto, 2009). Recently, a new way of administering chemotherapy was devised, called
"metronomic" which means frequent or daily administration of chemotherapeutic agents at doses significantly lower than maximum tolerated dose, with no long drug-free breaks
(Bertolini et al., 2003; Gasparini, 2001; Hanahan et al., 2000; Man et al., 2002). In addition to reduced levels of overt toxicity (Emmenegger et al., 2004), some preclinical models show superior anti-tumor effects and survival benefits over maximum tolerated dose (Man et al., 2002). This is because it has been shown that low dose metronomic chemotherapy targets endothelial cells of the growing tumor vasculature rather than cancer cells (Kerbel and Kamen, 2004; Man et al., 2002). Strikingly, the same studies showed that even tumors composed of cancer cells previously resistant to cyclophosphamide using conventional regimen responded to the same drug and completely regressed when treated with cyclophosphamide metronomically (Browder et al., 2000; Emmenegger et al., 2004).
Maximum tolerated dose cytotoxic chemotherapy also kills endothelial cells, but the difference is the absence of long breaks between drug administrations when metronomic dosing is employed. Due to this, there is significantly less opportunity for the repair of damaged endothelium and the antiangiogenic effects of chemotherapy irreversibly accumulate. In addition, Bertolini and colleges (Bertolini et al., 2003) showed that administration of cyclophosphamide at the maximum tolerated dose with drug-free breaks, caused mobilization of circulating EPC from the bone marrow a few days after administration making tumors more vascularized and drug resistant. On the contrary, administration of metronomic cyclophosphamide was associated with constant decrease in EPC with more durable inhibition of tumor growth.
13 In combination with antiangiogenic drugs or other types of targeted therapies, such as those specific for signaling transduction pathway molecules, metronomic chemotherapy might be more efficacious than used on its own (Colleoni et al., 2006; Daenen et al.,
2009; Dellapasqua et al., 2008; Garcia-Saenz et al., 2008; Jurado et al., 2008; Reardon et al., 2009).
Antiangiogenic Therapy
Tumor angiogenesis is an attractive therapeutic target, since it is shared by most
commonly occurring and perhaps all types of human cancers. In addition, clinical studies have suggested a direct correlation between the tumor blood vessel density and an
adverse prognosis in patients with a variety of solid tumors, including breast (Bosari et
al., 1992), lung (Macchiarini et al., 1992), prostate (Weidner et al., 1993), skin (Ruiter et
al., 1993) and head and neck tumors (Albo et al., 1994). Therefore, considering the
importance of vascular growth in tumor progression, therapeutic approaches targeting the tumor endothelium using antiangiogenic therapies, may provide a long-term and more
effective control of the disease compared to standard chemotherapeutic agents.
One of the advantages of using antiangiogenic agents is that under physiological
conditions, normal ECs are quiescent compared to tumor ECs that are actively
proliferating and migrating (Lin and Sessa, 2004). Therefore, the side effects of the short term administration of these inhibitors on normal tissues should be minimal (Klement et
al., 2000; Steeghs et al., 2007). Endothelial cells are also more genetically stable than
tumor cells (Kerbel, 1991). Their delivery to endothelial cells instead of cancer cells is not complicated by having to penetrate large bulky masses. Finally, successful
14 antiangiogenic agents may be applicable to many tumor types and may not be dependent on cancer cell type or growth fraction of cancer cells within a tumor (Albo et al., 1994).
The potential of antiangiogenic therapies lies is in their ability to 1) interfere with angiogenic ligands, their receptors or downstream signaling; 2) upregulate or deliver endogenous inhibitors or 3) directly affect the tumor vasculature (Carmeliet and Jain,
2000).
Although promising, the antiangiogenic therapies developed to date have their share of problems (Ebos et al, 2009). VEGF inhibitor therapies, such as bevacizumab, allow some patients to survive longer but have no effect on their overall survival (Hsu and
Wakelee, 2009). This could be due to the possible replacement of VEGF-A with other angiogenic pathways (Quesada et al., 2007). In addition, there may be selection and overgrowth of tumor-cell variants in hypoxic areas of the tumor that are less dependent on angiogenesis. In fact, in some cancer models, such as pancreatic neuroendocrine carcinoma and glioblastoma, treatment with multiple antiangiogenic inhibitors caused eventual tumor adaptation and even greater malignancy and invasiveness (Loges et al.,
2009; Paez-Ribes et al., 2009). Antiangiogenic therapies may also cause the remodeling of the tumor blood vessels into more mature, stabilized vessels that are less responsive to these drugs (Glade Bender et al., 2004; Jain, 2001).
Antiangiogenic therapies, such as bevacizumab, are generally well tolerated but some reports noted serious toxicities in patients. Commonly, bevacizumab is associated with gastrointestinal perforations and wound healing complications in about 2% of patients
(Shord et al., 2009; Simpkins et al., 2007). Patients over 65 years of age had higher risk of arterial thromboembolic complications while receiving bevacizumab (Ferrara et al.,
15 2005; Ranpura et al., 2010; Vaklavas et al., 2010; Zangari et al., 2009). Despite all these setbacks, antiangiogenic therapy alone or as recently suggested, in combination, is still one of the most promising approaches to cancer treatment (Gasparini et al, 2005; Lin and
Sessa, 2004; Pytel et al., 2009; Quesada et al., 2007; Steeghs et al., 2007).
Angiogenic Inhibitors in Combination with Chemotherapeutic Agents
Alternatively, much preclinical evidence indicates that combining antiangiogenic inhibitors with conventional MTD or metronomically administered chemotherapeutic agents results in additive or even synergistic anti-tumor effects (Gasparini et al., 2005).
This combination is thought to be especially effective using metronomic chemotherapy that would damage dividing ECs in tumor blood vessels, while antiangiogenic therapy would target key survival signals for ECs, all leading to subsequent killing of cancer cells
(Daenen et al., 2009). Based on this, in 2004, USA FDA approved bevacizumab
(Avastin) - a humanized monoclonal antibody against VEGF - for the treatment of metastatic colorectal cancer in combination with 5-fluorouracil-based chemotherapy regimens (Hurwitz et al., 2004). These approaches show promise, and the best way to evaluate the most effective combinations of antiangiogenic agents with chemotherapy or other biological agents is to test these combinations in vitro and in vivo models.
TIE2, ENDOTHELIAL TYROSINE KINASE RECEPTOR
Activation of endothelial receptor tyrosine kinases (RTKs), such as Tie2 and
VEGFR2 (to be introduced later), by binding of their corresponding ligands, plays a
16 major role in blood vessel differentiation and growth (Yancopoulos et al., 2000). Signal transduction pathways mediated by RTKs are initiated upon ligand binding, followed by receptor-mediated dimerization and autophosphorylation on specific tyrosine residues
(van der Geer et al., 1994; Schlessinger, 2000). Receptor cross-phosphorylation has two effects on receptor function - to further enhance the receptor's tyrosine kinase activity and to provide high affinity binding sites for many intracellular signaling molecules containing domains such as SH2 or PTB (Pawson and Scott, 1997; Schlessinger, 2000).
Other signaling molecules are recruited through a regulated cascade of protein-protein interactions that assemble into organized biochemical networks leading to the activation of important signaling pathways inside the cell (Jones and Dumont, 2000).
Tie2 and Angiopoietins - Function and Structure
The Tie receptors play an important role in angiogenic remodeling and vessel stabilization (Shalaby et al., 1995). Tiel and Tie2/Tek (tyrosine kinase with immunoglobulin and EGF homologous regions) belong to the family of tyrosine kinase receptors. Tie2 has a unique extracellular ligand-binding domain composed of immunoglobulin domains, epithelial growth factor and fibronectin-like 3 repeats. Thirty three percent of its extracellular domain and 76% of intracellular tyrosine kinase domain display similarity with Tiel (Davis et al., 2003; Jones and Dumont, 2000). Although
Tie2 was originally thought to be expressed exclusively on endothelial cells (Schnurch and Risau, 1993), Tie2 was also found on non-endothelial cells such as mural cells, keratinocytes, neutrophils, monocytes, synovial lining cells, eosinophils as well as
17 various carcinoma cells (Iurlaro et al., 2003; Makinde and Agrawal, 2008; Neagoe et al.,
2009; Shahrara et al., 2002; Venneri et al., 2007; Wolfram et al., 2009) .
Angiopoietins are a novel family of growth factors that bind specifically to Tie-2
(Hayes et al., 1999; Jones and Dumont, 2000; Valenzuela et al., 1999; Witzenbichler et al., 1998). The ligands angiopoietin-1 (Angl) and angiopoietin-2 (Ang2) both bind to
Tie2 with similar affinities but at different binding sites (Fukuhara et al.; Hayes et al.,
1999; Jones and Dumont, 2000; Witzenbichler et al., 1998). While Angl is considered an agonist, Ang2 signaling may be context dependent but is mostly known for its antagonistic properties (Maisonpierre et al., 1997; Yuan et al., 2009). Ang3 in mouse and
Ang4 in human represent interspecies orthologues and are more different than counterparts of Angl and Ang2 in mouse and human (Valenzuela et al., 1999).
In addition, Angiopoietin structural differences are correlated with their differences in function and expression. Once secreted, Angl gets incorporated into the extracellular matrix (Xu and Yu, 2001). Ang2 is not associated with the matrix but is instead stored in Weibel-Palade bodies in the cytoplasm of endothelial cells from where it can be rapidly secreted (Fiedler et al., 2004). Ang3 is tethered on the cell's surface via heparin sulphate proteoglycans. Also, while Angl is expressed by many different cell types in various tissues, Ang2 is mainly expressed in endothelial cells, primarily at the sites of angiogenesis. Angl and Ang2 expression may be modulated by stimulators such as IL-lp (Fan et al., 2004), TNF-a (Gravallese et al., 2003), VEGF (Makinde et al., 2006) and hypoxia (Oh et al., 1999). Although the function of Ang3 and Ang4 in angiogenesis is contraversial, its generally considered that Ang3 acts as antagonist by interfering with
Angl activation of Tie2 in tumor growth (Vanezuela et al, 1999). In mouse, Ang3 was
18 found to strongly activate Tie2 recepor, phenomena not observed in its human counterpart. Ang4 displayed no species selectivity (Lee et al, 2004). Ang4 phosphorylates
Tie2 in human endothelial cells (Vanezuela et al, 1999).
Angl-Tie2 signaling has an anti-apoptotic effect in endothelial cells
(Papapetropoulos et al., 2000). It also signals for localization of cell adhesion molecules in cell junctions and the recruitment of smooth muscle cells resulting in blood vessel stabilization (Fukuhara et al., 2010). Angl also has a chemotactic effect on endothelial cells but little or no effect on EC proliferation (Witzenbichler et al., 1998). Moreover,
Angl signals for differentiation of mesencymal cells into vascular smooth muscle cells in the embryo and is involved in maintenance of the quiescent mature vasculature in the adult (Iurlaro et al., 2003; Suri et al., 1996). In fact, Angl-Tie2 plays a crucial role in maintaining haematopoietic stem cells in their quiescent state in the bone marrow (Peters et al., 2004) (Eklund and Olsen, 2006; Jones and Dumont, 2000).
Lessons Learned From Knockout and Overexpression Studies
While Tie2 knock-out in mice resulted in embryonic lethality E9.5-12.5, closer observations revealed normal patterning of large vessels but severely affected vascular branching and differentiation of the microcirculation (Breier, 2000; Dumont et al., 1994;
Suri et al., 1996). Decreases in the number of endothelial-perivascular cell contacts in these embryos suggested that Tie2 may play a role in blood vessel maturation and stabilization (Lindahl et al., 1998). Mice deficient in Angl expression have a similar but less severe phenotype as Tie2-deficient mice, with impaired recruitment of perivascular cells and embryonic lethality at about El2.5. In contrast, mice overexpressing Angl have
19 highly branched vessels that are resistant to leakage (Thurston et al., 1999). Similar to
Angl and Tie2 knockouts, Ang2 overexpression was also embryonically lethal. In contrast, Ang2 deficient mice were born alive but had severe problems with lymphatic drainage (Dellinger et al., 2008; Gale et al., 2002; Shimoda et al., 2007). Collectively, these results illustrate that the levels of Tie2 signaling are critical to the establishment and maintenance of a normal vasculature. Disruption of this carefully orchestrated balance results in abnormalities in the cardiovascular system. Thus, Tie2 expression was found to be upregulated at the sites of active angiogenesis (cycling ovary and healing skin wounds) as well as vascular "hot spots" in cancer specimens (Peters et al., 2004).
Significance of Tie2 signaling transduction pathway
During angiogenesis, ECs proliferate, produce molecules that degrade ECM, change their adhesive properties, migrate, avoid apoptosis and differentiate into vascular tubes. These processes are controlled by signals received from their environment, that initiate signal transduction cascades eventually leading to changes in gene transcription that determines the behavior of the cell (Munoz-Chapuli et al., 2004). The importance of these pathways and their interrelationships is crucial for the development of new targets for antiangiogenic therapies.
Studies that disrupted Tie2 signaling pathways in mice show that activation of
Tie2 by angiopoietins appears to control at least two different signal transduction cascades: EC survival and EC migration necessary for angiogenic sprout formation
(Jones and Dumont, 2000). EC survival is regulated thorough the binding of Angl ligand to Tie2, inducing Tie2 phosphorylation (Hayes et al., 1999). Binding of p85 regulatory
20 subunit of phosphatidylinositol 3" (PI 3)-kinase to the phosphorylated Tie2 receptor, causes activation of PI3-kinase (Kim et al., 2000a), which in turn activates serine- threonine kinase Akt (also known as protein kinase B or PKB) (Downward, 1998). Akt then induces and controls anti-apoptotic signal transduction pathways (Jones and
Dumont, 2000). Specifically, activated Akt stimulates phosphorylation and inhibition of pro-apoptosis proteins like Bad, caspase-9, -7 and -3 (family of cysteine proteases that regulate apoptosis) (Adams and Cory, 2001), and an increase in survival and nitric oxide production (Papapetropoulos et al., 2000).
Mouse embryos lacking Tie2 signaling pathways displayed defective vessel sprouting and remodeling suggesting an inability of EC to alter their intracellular architecture and secrete MMPs that would allow them to migrate into surrounding basement membrane (Wilting and Christ, 1996). In vitro studies revealed that Angl stimulates EC migration and sprouting through the activation of PI3-kinase (Brkovic et al., 2007), that in turn induces tyrosine phosphorylation of the cytoskeletal regulatory protein focal adhesion kinase (FAK) and secretion of MMP-2 (Kim et al., 2000b).
During EC migration, phosphorylated Tie2 can also associate with signaling molecules such as the docking protein Dok-R (also called p56Dok2 and FRIP) that is involved in actin cytoskeleton reorganization and alters the shape and migratory properties of cells (Jones and Dumont, 1998).
Tie2 signaling may be regulated in several ways. Binding of Angl leads to Tie2 internalization and degradation (Bogdanovic et al., 2006). In un-stimulated cells, the half- life of Tie2 receptor is about 9h while Angl stimulation reduces the half-life to 3h
(Bogdanovic et al., 2006). At the same time, the rate of Tie2 synthesis does not change,
21 regardless of the presence of Angl. Once the activation of Tie2 is completed, Angl is not degraded but instead it is released back into the surrounding medium (Bogdanovic et al.,
2006).
Tie2 signaling can be inhibited by antagonists such as Ang2, in some cases Angl, and soluble Tie2 (sTie2Fc). sTie2Fc is a soluble form of extracellular domain of Tie2 present in the plasma of healthy individuals and may be a natural regulatory mechanism
(Harris et al., 2001). In fact, several stimulators such as VEGF and basic fibroblast growth factor have been implicated in "shedding" of Tie2 from the cell surface to form
75-kDa soluble Tie2 by proteolytic cleavage (Findley et al., 2007).
Role of Tie2 in Cancer
Up-regulation of Angl in tumor types such as high-grade gliomas, non-small cell lung carcinoma, ovarian, breast and gastric is highly correlated to tumor malignancy
(Chung et al., 2006; Giuliani et al., 2005; Hata et al., 2004; Holopainen et al., 2009;
Machein et al., 2004; Makinde and Agrawal, 2008; Shim et al., 2002; Takahama et al.,
1999; Torimura et al., 2004; Yamakawa et al., 2004; Zhang et al., 2006). On the contrary, Angl overexpression in breast, colon, Lewis lung carcinoma and squamous cell carcinoma cell lines, produced significant anti-tumor effects, possibly due to Angl mediated stabilization of tumor vasculature, making it resistant to angiogenic stimuli
(Tian et al., 2002). Ang2 is frequently unregulated in tumor vasculature and is associated with poor prognosis (Imanishi et al., 2007; Kunz et al., 2006; Moon et al., 2003;
Takanami, 2004; Tanaka et al., 1999; Torimura et al., 2004; Yu and Stamenkovic, 2001;
Zhang et al., 2003). These findings may help establish a role for the Tie2 pathway in
22 pathological angiogenesis, such as tumor angiogenesis, suggesting that modulation of
Tie2 signaling may have broad application in the treatment of cancer (Brown and
Giaccia, 1998).
Therapeutic manipulation of vascular growth in many vascular disease states may be possible by targeting specific signaling molecules in relevant pathways. Inhibition of signaling pathways involved in endothelial survival may be beneficial in diseases with excessive angiogenesis, such as cancer. In fact, some studies have already shown the effectiveness of angiogenesis inhibition in tumor-bearing mice using angiopoietin inhibitors (Brown et al., 2010). Therefore, although angiopoietin and/or Tie2 blockade may be an effective way to alter EC survival, other antiangiogenic agents and chemotherapeutic agents should be employed in combination with it, to achieve significant tumor regression.
In order to determine its full potential for cancer therapy, tumor specific effects on
Tie2 signaling need to be examined. On the other hand, an enrichment of signaling pathways that promote angiogenesis would help in diseases with insufficient angiogenesis such as ischemic or injured tissue (Jones and Dumont, 2000). Therefore, learning how to manipulate Tie2 signal transduction pathways holds great clinical potential for use of anti- and pro- angiogenic therapies.
23 VEGF PATHWAY IN TUMOR ANGIOGENESIS
Vascular Endothelial Growth Factor
VEGF (vascular endothelial growth factor) is an endothelial specific growth factor and main regulator of both normal and pathological angiogenesis (Ferrara, 2000).
Binding of VEGF to its corresponding receptors signals endothelial cell survival, mitogenesis, migration, differentiation, vascular permeability and mobilization of endothelial progenitor cells from bone marrow into peripheral circulation (Abedi and
Zachary, 1997; Asahara et al., 1999; Barleon et al., 1996; Carmeliet et al., 1996; Gerber et al., 1998; Millauer et al., 1993; Neufeld et al., 1999). Homozygous or heterozygous deletion of VEGF gene in mice resulted in embryonic lethality with defects in vasculogenesis and cardiovascular abnormalities, demonstrating its significance in development (Carmeliet et al., 1996; Ferrara et al., 2003).
VEGF growth factor family is composed of seven secreted glycoproteins -
VEGF-A (commonly referred to as VEGF), VEGF-B, VEGF-C, VEGFD, VEGF-E,
VEGF-F (found in snake venom)(Yamazaki et al., 2005) and placental growth factor
(PlGF)-l and -2 (Houck et al., 1991; Park et al., 1994; Park et al., 1993). VEGF-A gene undergoes alternative splicing to yield mature isoforms of 121, 165, 189, and 206 amino acids (one amino acid less in mice). Less frequent variants such as VEGF145 and VEGF183 have also been reported (Houck et al., 1991; Poltorak et al., 1997). VEGF121 is freely secreted; VEGF]89 and VEGF206 are sequestered in the ECM and require cleavage by proteases for their activation (Dvorak, 2002) while VEGF165 exists in both soluble and
ECM-bond form (Ferrara et al., 2003; Keyt et al., 1996; Neufeld et al., 1999). ECM-
24 bound isoforms of VEGF-A, VEGF-C and VEGF-D can be released by plasmin cleavage at the C-terminus or by MMP-9, generating bioactive fragment (Olofsson et al., 1998;
Park et al., 1993). In adults, VEGF-A plays an important role in normal processes such as wound healing (Kumar et al., 2009), ovulation (Robinson et al., 2009), menstruation
(Punyadeera et al., 2006), blood pressure maintenance (Langenberg et al., 2009) and pregnancy (Brown et al., 1992), as well as pathological conditions such as psoriasis
(Detmar et al., 1994), macular degeneration (Browning et al., 2008) diabetic retinopathy
(Caldwell et al., 2003) etc.
VEGF, in particular VEGFI65 isoform, is commonly over-expressed in the majority of human solid tumors, such as colon cancer (Takahashi et al., 1995), gastrointestinal cancer (Takahashi et al., 2003), pancreatic cancer (Ikeda et al., 1999), breast cancer (Berns et al., 2003), prostate cancer (George et al., 2001), lung cancer
(Fontanini et al., 1998; Fontanini et al., 1997a; Fontanini et al., 1997b) and melanoma
(Potgens et al., 1995). This overexpression in melanoma is usually associated with tumor progression and poor prognosis (Gorski et al., 2003). In fact, early stage primary human cutaneous melanoma is known to remain relatively dormant and avascular for up to a decade with minimal production of VEGF (Gitay-Goren et al., 1993). VEGF, not normally produced by normal melanocytes, is upregulated during melanoma progression, while hyperpermeability of the newly formed blood vessels caused by VEGF, plays an important role in metastasis (Salven et al., 1997).
25 Signaling through Vascular Endothelial Growth Factor Receptors
VEGF functions mainly in a paracrine and to a lesser extend autocrine fashion, by binding specific receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1), present on endothelial cells and/or some tumor cells (Ferrara et al., 2003; Neufeld et al., 1999). All of the VEGF-A iso forms bind to both VEGFR1 and VEGFR2, while P1GF-1, P1GF-2 and
VEGF-B are specific for VEGFR1 activation. VEGFR3 binds VEGF-C and VEGF-D
(Kaipainen et al., 1995) and has been found to be primarily associated with lymphangiogenesis (Paavonen et al., 2000). VEGFR3 is activated and upregulated in breast cancer and melanoma, with elevated levels of VEGF-C or VEGF-D associated with lymph node metastasis in patients.
VEGFR1 and VEGFR2 consist of 1338 and 1356 amino acids in humans, respectively that consist of extracellular ligand-binding domain, transmembrane domain, tyrosine kinase domain and downstream carboxyl terminal region (Millauer et al., 1993;
Shibuya et al., 1990). VEGFR1 and VEGFR2 are highly homologous to each other in overall structure (Shibuya et al., 1990). VEGFR2 is mostly expressed on endothelial cells but may also be expressed in lower levels in hematopoietic cells (Asahara et al., 1999;
Lyden et al., 2001), neuronal cells, osteoblasts, pancreatic duct cells, retinal progenitor cells and megakaryocyates (Matsumoto and Mugishima, 2006). The biological role of
VEGFR2 in these non-endothelial cells still remains to be clarified.
VEGFR2 is responsible for the majority of downstream effects of VEGF-A signaling, including endothelial permeability, proliferation, invasion, migration and survival (Ferrara et al., 2003). In fact, the ability of VEGF to enhance microvascular permeability through VEGFR2 is its one of its most important properties, in particular
26 with regards to hyperpermeability of tumor vessels due to its overexpression (Dvorak,
2002). Recent evidence suggests that this permeability change involves signaling though
NO production and activation of Akt, in addition to activation of Erkl/2 pathway though prostaglandin PG12 (Bates and Harper, 2002). Endothelial proliferation mediated though
VEGFR2 potentially involves two pathways - activation of Erkl/2 and JNK/SAPK
(members of the MAPK family) and/or protein kinase C pathway regulated by
NO(Zachary and Gliki, 2001). Endothelial survival and migration involves activation of the P13K-Akt pathway and FAK (focal adhesion kinase), respectively (Abedi and
Zachary, 1997; Gerber et al., 1998).
The precise function of VEGFR1 is still not clear. It is a receptor for VEGF-A (de
Vries et al., 1992) with an ability to bind VEGF-B and P1GF. Mouse embryos lacking
VEGFR1 die in utero between days 8.5 to 9.5 due to excessive hemangioblast proliferation and poor organization of vascular structures (Fong et al., 1995). This receptor was initially thought to be a negative regulator of VEGF activity either by acting as a decoy receptor for VEGF or by downregulating VEGFR2 signaling (Backer et al.,
2009). In fact, a repressor motif has been identified in the juxtamembrane region of
VEGFR1 that impairs PI3K signaling and endothelial cell migration in response to VEGF stimulation (Shibuya et al., 1990). Alternatively, studies have shown that VEGFR1 is a potent, positive regulator of tumor angiogenesis (Hiratsuka et al., 2001) participating in monocyte migration (Kaplan et al., 2005), recruitment of endothelial progenitors (Kaplan et al., 2005; Nolan et al., 2007), increasing adhesive properties of natural killer cells
(Chen et al., 2002) and inducing growth factors from liver sinusoidal endothelial cells
(LeCouter et al., 2003).
27 Recent Developments in VEGFR2 Targeted Therapies
Various approaches have been taken to interfere with the VEGF/VEGFR system as an antiangiogenic therapy, including antagonistic antibodies (Hsu and Wakelee, 2009), recombinant soluble VEGFR proteins (Lin et al., 1998b) and small molecule tyrosine kinase inhibitors (Backer et al., 2009).
Bevacizumab (Avastin) is a recombinant humanized monoclonal antibody that binds to VEGF-A and its isoforms. It was the first antiangiogenic drug to be approved by the USA FDA in combination with 5-fluorouracil for treatment of metastatic colorectal cancer (Hurwitz et al., 2004). Administration of bevacizumab is usually well-tolerated with toxicities such as hypertension, proteinuria, minor and major hemorrhage, arterial and venous thrombosis, wound healing complications and gastrointestinal perforation
(Cohen et al., 2007; Miller et al., 2007). Bevacizumab inhibits VEGF-induced endothelial cell proliferation, tumor angiogenesis and tumor growth (Ferrara et al., 2005). Treatment with VEGF inhibiting drugs was associated with normalization of blood vessels, facilitating delivery of other drugs to the tumor (Jain, 2001).
Preclinical studies using anti-VEGFR2 antibodies demonstrated decreased VEGF- induced signaling, decreased angiogenesis and decreased primary and metastatic growth in variety of tumors (Ran et al., 2003; Zhang et al., 2004). Several small molecule inhibitors of VEGFR tyrosine kinase activity have been developed that also have an ability to affect kinase activity of basic FGFR, EGFR, PDGFRa and PDGFRp, c-kit and
VEGFR3 (Backer et al., 2009; Steeghs et al., 2007).
Critical cell functions such as growth, differentiation, angiogenesis, and inhibition of apoptosis are controlled through receptor tyrosine kinases (Rrause and Van Etten,
28 2005). In malignancies, these signaling pathways are often exploited to optimize tumor growth and metastasis and therefore represent attractive targets for cancer therapy.
Developed recently, small-molecule multiple-kinase inhibitors (Gerber, 2008; Liao and
Andrews, 2007; Richter and Zhang, 2005; Smith et al., 2004) hinder phosphorylation of several membrane receptor protein kinases such as VEGFR, PDGFR, the human EGFR family, and cytoplasmic receptors such as c-Kit, Raf kinase, and FLT3. Like bevacizumab, some of these multiple-kinase inhibitors - sorafenib/BAY 43-9006
(Hartmann et al., 2009; Ivy et al., 2009; Kim et al., 2009; Miyanaga and Akaza, 2009), sunitinib/SUl 1248 (Cabebe and Wakelee, 2006) and vatalanib/ PTK787/ZK222584
(Scott et al., 2007) have been studied in clinical trials with favorable toxicity reports and encouraging results in treatment of advanced colorectal, renal cell, breast and non-small cell lung cancers (NSCLC) (de Castro Junior et al., 2006).
Sunitinib is a novel, selective, small molecule inhibitor of multiple RTKs, such as
PDGFRa and PDGFRp, VEGFR 1, VEGFR2 and VEGFR3, stem cell factor receptor
(KIT), Fms-like tyrosine kinase-3 (FLT3), colony stimulating factor receptor Type 1
(CSF-1R) and glial cell-line derived neurotrophic factor receptor (RET) (Atkins et al.,
2006; Cabebe and Wakelee, 2006; Hartmann et al., 2009; Kim et al., 2009; Mendel et al.,
2003; Steeghs et al., 2007; Sun et al., 2003). Clinical trials show that sunitinib was highly efficacious in regression and growth arrest, substantially reducing growth of various established human (colorectal: HT-29, skin: A431, colon: Colo205, lung: H-460, melanoma: A375, and breast: MDA-MB-435) or rat (brain: C6) preclinical tumor models with acceptable toxicities (Abrams et al., 2003a; Abrams et al., 2003b; Mendel et al.,
2003). In fact, in January 2006, sunitinib was approved by the USA FDA for the
29 treatment of gastrointestinal stromal tumors (Bond et al., 2009; Schoffski et al., 2009) and advanced renal-cell carcinoma (Ivy et al., 2009; Le Tourneau et al., 2007; Michels et al., 2009; Takahashi et al., 2003). Although the development of sunitinib and the other
VEGF-targeted agents gives cause for optimism and represents a triumph in the translation of basic biology into clinical efficacy, much research is still needed to fully realize its promise (Atkins et al., 2006).
THE ROLE OF THROMBOSPONDIN-1 IN CANCER
Thrombospondin-1 (TSP-1), a potent endogenous inhibitor of angiogenesis, is a member of a multigene family of secreted, high-molecular weight, multifunctional extracellular matrix glycoproteins involved in wide variety of cellular functions and interactions (Chen et al., 2000). TSP-1 is synthesized by blood platelets, endothelial cells, fibroblasts, smooth muscle cells and keratinocytes (Carlson et al., 2008). In cancer, through its influence on cell-matrix interactions and angiogenesis, TSP-1 can act as an endogenous regulator of tumor growth and metastasis (Ren et al., 2006). Additionally,
Rofstad and Graff showed that growth of metastatic tumor cells is inhibited by TSP-1
(Rofstad et al., 2004).
TSP-1 has been associated with both a supportive (Guo et al., 1992; Taraboletti et al., 2000; Wang et al., 1996) and inhibitory (Lawler, 2002; Nor et al., 2000; Rodriguez-
Manzaneque et al., 2001; Rofstad et al., 2004) role in tumor invasiveness and progression. Furthermore, these studies have shown that TSP-1 might either induce or
30 suppress angiogenesis, depending on which domain of the molecule is active and/or available, or whether different TSP-1 receptors are present on endothelial cells.
TSP-1 is also highly expressed in many human cancers, such as breast, colon,
cervical, pancreatic and ovarian carcinoma and its increased expression can be detected
in the serum of cancer patients (Qian and Tuszynski, 1996). Human melanoma cell lines have also been shown to synthesize and secrete TSP-1 (Apelgren and Bumol, 1989) but
the expression/secretion varied among the cell lines examined (Varani et al., 1989). The two major mechanisms by which altered TSP-1 expression is likely to influence
melanoma progression and metastasis are via angiogenesis, and melanoma-ECM
interactions. TSP-1 inhibits angiogenesis (de Fraipont et al., 2001) by blocking
endothelial cell migration and by induction of endothelial cell apoptosis. This is achieved
through binding of TSP-1 to its receptor, CD36, which was shown to be an essential
mediator of antiangiogenic action of TSP-1 on endothelial cells in vitro (Dawson et al.,
1997; Greenwalt et al., 1992; Silverstein and Febbraio, 2009). CD36, also called
glycoprotein IV, is an 88-kDa transmembrane glycoprotein found on platelets, monocytes
and microvascular endothelial cells, and is an adhesion receptor for TSP-1. Jimenez et al.,
have shown that TSP-1 activation of the CD36-Fyn-caspase-3-p38 MAPK cascade is
essential for TSP-1 's antiangiogenic effect and for induction of endothelial cell apoptosis
(Jimenez et al., 2000).
Also, wild-type p53 positively regulates TSP-1 gene promoter sequences and
inhibits angiogenesis through upregulation of TSP-1 synthesis in malignant melanoma
(Grant et al., 1998). Mutations in p53 gene are relatively infrequent in primary
melanomas (<20%), but when they do occur, reduced expression of TSP-1 causes
31 increased angiogenesis and metastatic progression (Dameron et al., 1994). In addition, if
TSP-1 is strongly expressed in tumor stroma (significant amounts of which can be present in 43% of melanomas), this correlates with increased proliferation and microvascular density and therefore, decreased patient survival (Straume and Akslen,
2001; Trotter et al., 2003). Stromal TSP-1 presumably enhances melanoma progression through its role in melanoma cell-matrix interactions. Increase in the expression of avP3 integrin, associated with melanoma progression and metastatic potential has been reported in both melanoma cell lines as well as clinical specimens (Seftor et al., 1992).
TSP-1 modulates avP3 function through integrin-associate protein and promotes spreading of melanoma cells on vitronectin (Chandrasekaran et al., 2000; Gao et al, 1996).
Besides inhibiting angiogenesis directly by interfering with endothelial cell migration and apopotosis, TSP-1 can also inhibit angiogenesis indirectly by affecting
VEGF bioavailability. This can be achieved though TSP-1 inhibition of the activation of matrix metalloproteinase 9, thus suppressing the release of stored VEGF from the extracellular matrix (Rodriguez-Manzaneque et al., 2001). In addition, TSP-1 can bind directly to VEGF and sequester it (Kerbel and Kamen, 2004).
AUTOCRINE AND INTRACRINE SIGNALING OF RECEPTOR TYROSINE
KINASES
It has been suggested that some extracellular signaling peptides such as hormones, growth factors etc. play a role in both autocrine and intracrine signaling. Autocrine signaling is defined as a type of cells signaling in which a cell produces a growth factor
32 to which it also responds (Shvartsman et al, 2002). Unfortunately, cancer cells often use autocrine signaling to overcome normal controls on cell proliferation and survival. By secreting signals that act back on the cell's own receptors, cancer cells can stimulate their own survival and proliferation and therefore survive and proliferate in places where normal cells of the same type would not (Iqbal et al, 2010). Studies have shown that
VEGF acts as an autocrine growth and survival factor for VEGF receptor expressing tumor cells (Bachelder et al, 2001; Fan et al, 2005; Vincent et al, 2005; Sher et al, 2009).
Secreted factors and cell surface receptors can also be internalized by endocytosis and translocated to the cytoplasm. Once in the cytoplasm, they can be recycled, degraded or translocated to the nucleus if the secreted factor or receptor being internalized has nuclear locations signals (NLS) (Blazquez et al, 2006). In the nucleus, these molecules have an ability to regulate transcription of specific genes by direct binding to the transcription factors (Planque, 2006). For instance, phosphorylated endothelial growth factor receptor (EGFR) undergoes nuclear translocation where it induces transcription of genes essential for cell proliferation and cell-cycle regulation (Duttmann et al, 2010).
Intracrine signaling can occur in one of the following ways: (1) by binding of growth factor to the receptor within the secretory pathway prior to surface localization or (2) by being secreted and binding to its RTK on the same cell to signal intracellularly in the internalized endosome (Re & Cook, 2008). Intracrine signaling is commonly observed in cancer growth and in endothelial cell proliferation during angiogenesis. In fact, Lee et al. have shown that an internal VEGF/VEGFR1 signaling loop in breast cancer gives survival advantage to these cells (Lee et al. 2007).
33 In 2000, Li et al. showed for the first time that VEGF traffics to the nucleus and can act as an intracrine signal in endothelial cells (Li & Keller, 2000). In fact, using an in vitro wounding model, they demonstrated that in cells away from the wound area, VEGF gets internalized upon binding to the VEGFR2 and this complex was transported to endosomes, whereas in the cells facing the wound, VEGF/VEGFR2 complex accumulated in the nucleus. This accumulation in the nucleus resulted in the increased expression of different wound healing proteins. Subsequently, intracrine VEGF signaling was demonstrated in bone marrow stem cells and some cancer cells (Gerber et al, 2002;
Santos & Dias, 2004). In addition to the VEGF, after phosphorylation, VEGFR2 receptor has been reported to internalize inside the cytoplasm and translocate into the nucleus of endothelial cells (Feng et al, 1999; Blazquez et al, 2006).
34 RATIONALE
Receptor tyrosine kinases play some of the most crucial roles for the survival and maintenance of endothelial cells. It is therefore not surprising for these receptors to be at the forefront of current research in the field of cancer therapeutics. Various molecules targeting different parts of these receptors (extracellular domain, ligand binding domain, kinase domain, etc.) are in current clinical trials. Moreover, these tyrosine kinase inhibitors are also being evaluated alone as well as in combinations in order to circumvent one of the most common occurrences with cancer therapies - acquired resistance.
During the course of these studies, we found the expression of tyrosine kinase receptor, VEGFR2, by our melanoma cells themselves. For our first objective, studies were designed to determine the existence of autocrine and intracrine VEGFR2 signaling loops in these melanoma cell lines and their possible functions through the inhibition of
VEGF/VEGFR2 pathway both in vivo and in vitro using anti-VEGF neutralizing antibody and small molecule VEGFR2 specific inhibitor, ZM323881. If fact, we hypothesized that simultaneous blockage of VEGF/VEGFR2 on both endothelial and cancer cells would give us superior antiangiogenic and potent anti-tumor effects.
Besides these specifically targeted antiangiogenic molecular inhibitors, researchers have found a way to utilize some of the old and standard cytotoxic chemotherapeutic agents in a new way - administering them at dosages of up to ten times below maximum tolerated dose normally used in patient treatments and as such giving them to the patients on a regular basis, avoiding the potential side-effects of maximum tolerated doses.
35 Our laboratory focuses on studies of another tyrosine kinase receptor, Tie2, and its expression in cancer. For the second objective, studies in this thesis were specifically designed to look more closely into Tie2 expression in melanoma xenografted tumors. We hypothesized that Tie2 inhibition will have an anti-tumor effect both when administered alone and in combination with metronomic low dose cyclophosphamide chemotherapy.
This chemotherapy has been shown to be cytotoxic to angiogenic cells, providing antiangiogenic response, as well as to function indirectly through a different antiangiogenic molecule, thrombospondin-1. We also wanted to determine the impact of
Tie2-negative blood vessels, shown to exist in about 15% of the tumor blood vessels in melanoma, on the efficacy of both Tie2 targeted and antiangiogenic, non-targeted therapies.
Since the vessels in the tumors are not only formed from pre-existing capillaries by the process of angiogenesis but also by the aid of bone-marrow derived endothelial progenitor cells, for the third objective, we isolated these cells and studied the endothelial cells they differentiated into, with respect to their expression of Tie2 and VEGFR2 receptors. In fact, these endothelial, EPC-derived cells, showed variable Tie2 and
VEGFR2 expression. We hypothesized that the functional differences, if any, between these clones will have an effect on their ability to incorporate into blood vessels when injected into nude mice with subcutaneous Matrigel plugs.
In conclusion, all these studies look into various aspects of cancer angiogenesis, primarily to determine in more detail the functioning of Tie2 and VEGFR2 in endothelial cells as well as melanoma cells.
36 CHAPTER 2 - Functional autocrine and intracrine VEGF/VEGFR2 signaling loop in malignant melanoma
37 INTRODUCTION
Secretion of pro-angiogenic factors during tumor angiogenesis is crucial for tumor growth and metastasis. VEGF (VEGF-A) or vascular endothelial growth factor, is one of the main pro-angiogenic regulators of both normal and pathological angiogenesis
(Ferrara, 2002). To date, bevacizumab or Avastin, anti-VEGF antibody, alone or in combination with chemotherapy has shown clinical activity (Ferrara et al., 2005) in colorectal (Hurwitz, 2004; Hurwitz et al, 2004), breast (Dellapasqua et al., 2008; Miller et al., 2007; Wedam et al., 2006), glioma (Reardon et al., 2009), ovarian (Jurado et al.,
2008), non-small cell lung (Cohen et al., 2007) and metastatic renal cell carcinoma (Yang et al., 2003), validating VEGF pathway inhibitors as an important new treatment modality in cancer therapy (McCarthy, 2003).
Within solid tumors, VEGF is mainly produced by tumor cells and it binds in paracrine fashion to VEGFR1 (Flt-1), VEGFR2 (KDR, human/Flk-1, mouse) and neuropilin receptors (NP-1 and NP-2) (Neufeld et al., 1999). In the cell, VEGFR2 is produced as a 150-kDa molecule that is further glycosylated to a protein of about 200- kDa. Further glycosylation produces a 230-kDa form that is expressed on the cell surface
(Takahashi and Shibuya, 1997). VEGFR2 is responsible for the majority of downstream effects of VEGF including changes in vascular permeability, endothelial proliferation, invasion, migration and survival (Zachary and Gliki, 2001). Ligand-induced phosphorylation of VEGFR2 occurs at tyrosine residues 951, 1054, 1175 and 1214, where Y1175 and Y1214 are two major auto-phosphorylation sites of VEGFR2, with only Y1175 auto-phosphorylation leading to VEGF-dependent endothelial cell
38 proliferation (Matsumoto and Mugishima, 2006; Takahashi et al., 2001). Endothelial cell migration towards a gradient of VEGF is due to the phosphorylation of Y951 residue on
VEGFR2, which then recruits the adapter molecule TSAd resulting in stimulation of the migration signal (Matsumoto et al., 2005; Zeng et al., 2001b). Binding of VEGF to
VEGFR2 also activates downstream survival and migration pathways involving PI3- kinase-Akt and focal adhesion kinase (FAK), respectively (Zeng et al., 2001b).
In addition to these paracrine functions, tumor-derived VEGF may also be involved in autocrine stimulation of tumor growth, binding specifically to VEGFR1 and
VEGFR2 present on cancer cells themselves (Aesoy et al., 2008; Neuchrist et al., 2001;
Xia et al, 2006; Zhang et al., 2004). The presence of VEGF receptors on human melanoma cells suggests the possibility of an autocrine VEGF/VEGFR signaling loop in this disease (Graells et al., 2004; Lacal et al., 2005; Lazar-Molnar et al., 2000). Similarly,
Graells et al. in 2004 showed that overexpression of VEGFi65 in a melanoma cell line that expresses VEGFR2, favors cell growth and survival through MAPK and PI3K signaling pathways. In addition, some VEGF receptors may not be expressed on the surface of the tumor cells but instead within the cell, promoting survival through a VEGF/VEGFR
"intracrine" mechanism as seen with VEGFR1 in breast cancer cells (Lee et al., 2007) and hematopoietic stem cells (Gerber and Ferrara, 2005).
In locally advanced solid tumors, oxygen delivery is frequently reduced or even abolished due to abnormalities of the tumor vasculature. Up to 50-60% of locally advanced solid tumors may exhibit hypoxic and/or anoxic tissue areas that are heterogeneously distributed within the tumor mass (Vaupel et al., 1989). Hypoxia- induced alteration in gene expression may promote tumor progression via mechanisms
39 enabling cells to overcome nutritive deprivation, to escape from the hostile metabolic microenvironment and to favor unrestricted growth. Sustained hypoxia may thus lead to cellular changes resulting in a more clinically aggressive phenotype (Graeber et al.,
1996). Hypoxia plays an important role in regulation of VEGF expression (Detmar et al.,
1997). During hypoxia, HIF-1 alpha dimerizes with HIF-1 beta, and this complex translocates to the nucleus and binds to VEGF promoter, leading to increased VEGF transcription and therefore increased production of VEGF (Hicklin and Ellis, 2005). It is not uncommon for metastatic melanoma to contain hypoxic areas arising in the tissue distant from the blood supply (Vaupel et al., 1989).
We employed paired human melanoma cell lines (WM115 and WM239) derived from the same patient but from primary and metastatic lesions to investigate differences in expression of VEGF and VEGFR2 and evaluate the effects of hypoxia on VEGFR2 levels. Differences and functionality of VEGF and VEGFR2 was evaluated in vitro and in vivo using the anti-VEGF antibody bevacizumab alone or in combination with anti-
VEGFR2 small molecule inhibitor ZM323881 (Whittles et al., 2002). These studies identified functional autocrine as well as intracrine VEGF/VEGFR2 signaling in primary and metastatic melanoma cell lines and investigated the underlying differences in downstream signaling of these pathways, and their possible impact on tumor responses to
VEGF targeted therapy.
40 MATERIAL AND METHODS
A list of all chemicals and suppliers as well as the description of materials and solutions prepared for the following experiments are detailed in Appendices I and II, respectively.
Cell lines and culture conditions
The following cell lines were purchased from American Type Culture Collection and used in experiments - WM115 (primary melanoma (Rodeck et al., 1987)), WM239
(metastatic melanoma, isolated from a secondary lesion from the same patient (Rodeck et al., 1987)), bEnd3 (a mouse brain-derived polyoma middle T antigen transformed endothelial cell line), and 293T (human fetal kidney) (Graham et al., 1977). Primary bovine aortic endothelial cells (BAEC) were isolated from aorta of adult cattle and characterized as previously reported (Coomber, 1995). Cells were routinely cultured in
DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Gibco), sodium pyruvate (Sigma-Aldrich) and gentamicin (Gibco) at 37°C in 5% CO? and 95% air.
Establishment of tumor xenografts
All procedures described below were done according to the guidelines and recommendations of the Canadian Council of Animal Care and approved by the
University of Guelph Local Animal Care Committee. Tumor xenografts were established in female athymic nude mice (Charles River Canada) using human melanoma cell lines
WM115 and WM239. 2xl06 melanoma cells in 100 of 0.1% BSA were subcutaneously injected into the right flank of mice. Tumor growth was measured twice weekly using calipers and tumor size was calculated using the equation: volume = length
41 x width2 x 0.5. Once tumors reached approximately 100 mm3 in size, mice were randomly allocated into control or treatment group, 8 per group. Groups were treated for
14 (for WM115 xenografts) or 21 (for WM239 xenografts) days with bevacizumab as
Avastin™ i.p, 5|ig/kg twice weekly; mice in control groups received 100 PBS twice weekly. Mice were euthanized by CO2 asphyxiation followed by cervical dislocation.
Tumors were removed from the surrounding tissue, embedded in OCT cryomatrix and snap frozen in liquid nitrogen, fixed in 4% paraformaldehyde (USB Corporation) for 24h, or snap frozen in liquid nitrogen for future protein and RNA isolation and stored at -80°C.
Reverse transcription/polymerase chain reaction (RT-PCR)
WM115 and WM239, 293T and bENd3 cells were washed twice in phosphate- buffered saline (PBS) and lysed using 1.0 ml TriPure according to manufacture's conditions. Total RNA was quantified using Nano-drop analysis (Thermo Scientific) and
5 |ig of RNA was used for cDNA synthesis using the Turbo DNAse kit (Amgen). Primers specific for VEGFR2 were used to perform RT-PCR. The amplification was carried out at a cycle program for 30 cycles of 95°C for 1 min, 60°C for 1 mm and 72°C for 2 mm.
The PCR products were separated using 2% agarose gel, stained with ethidium bromide and revealed under UV light. Primer sequences for human and mouse VEGFR2 were 5'-
CCAAGAACTCCATGCCCTTA-3' for the anti-sense and 5'-
ATCCCTGTGGATCTGAAACG-3' for the sense strand. Negative controls were performed on samples containing no reverse transcriptase (RT) enzyme and using reaction buffer without cDNA template.
42 Western blotting
Cultured cell lines (WM115, WM239 and BAECs) were seeded at the appropriate densities and allowed to attach overnight in DMEM (Sigma-Aldrich) supplied with 10%
FBS (Gibco). Next day, cells were washed with PBS (Sigma-Aldrich) and serum-starved for 48 hours. To be certain that phosphorylation of VEGFR2 is achieved by expression of the VEGF ligand by melanoma cells itself and not by VEGF supplied in culture medium and added serum, we thoroughly washed cells in PBS and serum-starved them for 48h.
Following that, for some experiments melanoma cells were treated with control human
IgG antibody, 150 ng/ml of bevacizumab as Avastin™, anti-VEGF antibody and/or anti-
VEGFR2 small molecule inhibitor, ZM323881 (Tocris).
For experiments involving hypoxic conditions, cells were incubated in a Modular
Hypoxic Chamber (Billups-Rothenberg) under less than 0.1% oxygen for 24 and 48h.
Then, cells were lysed in protein lysis buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, ImM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta- glycerophosphate, 1 mM Na3V04, 1 (ig/ml leupeptin (Cell Signaling Technology), 1 mM
PMSF, 2 (ig/ml aprotinine and phosphatase inhibitor cocktail 2, according to manufacturer's instructions (Sigma-Aldrich)]. For experiments involving phosphorylated-protein detection, cells were treated with ImM pervanadate for 30 minutes prior to lysis. For separation of nuclear and cytosolic fractions, Nuclear and
Cytosolic Fractionation kit was used according to manufacturer's instructions
(LabVision).
43 Frozen tumor pieces from WM115 and WM239 xenografts were disrupted in freshly prepared lysis buffer (Cell Signaling) on ice. Tissues were further lysed on ice for
15 minutes and centrifuged at 16,000 x g for 20 minutes at 4 °C.
Total protein was determined using the Bio-rad Protein Assay (Bio-Rad,
Mississauga, ON), based on the method of Lowry (Lowry et al., 1951). The lysates were electrophoretically resolved on 7.5% polyacrylamide gels and transferred to a PVDF membrane (Roche). Membranes were first blocked for lh at room temperature in 5% milk or 5% BSA (Jackson ImmunoResearch) in TBS/Triton followed by overnight incubation at 4°C in primary antibodies diluted in 5% milk or 5% BSA in TBS/Triton.
Primary antibodies used were rabbit anti-VEGFR2, rabbit anti-phospho-VEGFR2
Tyr951, rabbit anti-phospho-VEGFR2 Tyrll75, rabbit anti-phospho-FAK, rabbit anti-
FAK, rabbit anti-phospho-Akt, rabbit pan Akt, rabbit anti-phospho p44/42 (all 1:1000 dilution, all Cell Signaling Technology), mouse anti-a-tubulin (1:400,000, Sigma-
Aldrich) and rabbit anti-lamin (1:1000, Cell Signaling Technology). Next day, membranes were washed and treated with appropriate POD conjugated secondary antibody (anti-rabbit POD at 1:10,000 and anti-mouse POD at 1:40,000 (Sigma-
Aldrich)). Immuno-complexes were visualized using Chemilluminescence detection kit
(Roche), exposed to X-ray film and quantified on a FluorChem 9900 gel documentation imaging system (Alpha Immunotech). Protein loading was normalized using a-tubulin bands for protein lysates.
44 Measurement of human VEGF protein levels in melanoma cell conditioned medium
A commercially available human VEGF ELISA kit (R&D Systems, Inc.,
Minneapolis, MN) was used to quantify VEGF in conditioned medium from WM115 and
WM239 cell lines. Briefly, 250,000 cells were plated in 6 well plates and allowed to attach overnight. Next day, cells were washed in PBS and medium was replaced with
DMEM containing 2% FBS. Following that, cells were treated with control human IgG antibody, 50 and 150 fig/ml of bevacizumab as Avastin™. Conditioned medium was collected 6 and 24h later and centrifuged to remove any floating cells. Conditioned medium was either used immediately or stored at 4°C until VEGF quantification.
Adherent cells were trypsinized and counted using a haemocytometer for normalization purposes.
In addition, VEGF ELISA kit (R&D Systems) was also used to quantify VEGF levels in lysed tumor xenografts from each of the treatment groups (control and bevacizumab). Briefly, frozen tumor pieces (3 per group) were defrosted on ice and lysed using manual grinder and cell lysis buffer (Cell Signaling). Protein was collected as previously described and VEGF ELISA performed as suggested in manufacturer's instructions.
Immunofluorescence for phosphorylated and native VEGFR2 receptor in cultured
cells
WM115 and WM239 cells were cultured on sterile glass cover slips for 48 hours and fixed with 4% paraformaldehyde (USB Corporation) at room temperature for 12 minutes. They were then washed with PBS with 0.3% Triton X-100 and non-specific
45 binding was blocked using serum free protein block (Dako) for 30 minutes. Cells were incubated in rabbit anti-VEGFR2 antibody (Cell Signaling Technology) diluted 1:50 in
PBS with 0.3% Triton X-100 overnight at 4°C or rabbit phospho-VEGFR2 Tyrl 175 (Cell
Signaling Technology) at the dilution of 1:400. Next, coverslips were washed in PBS and incubated with fluorescently tagged anti-rabbit Cy3 secondary antibody (1:200;
Jackson ImmunoResearch) for 30 minutes. Slides were then washed with PBS, stained with 4 ng/ml of DAPI (Dako) in PBS and mounted using fluorescent mounting medium
(Dako). Images were captured with a 20X objective using QCapture software calibrated to a Leica DMLB microscope fitted with a Q imaging QIC AM fast 13 94 digital camera.
Images were merged using Adobe Photoshop 7.0 (Adobe).
VEGFR2 immunostaining & blood vessel density quantification
To evaluate VEGFR2 expression patterns as well as determine blood vessel density, two blocks from each tumor xenograft were assessed. Cryosections, 8 ^m thick, were cut using a cryostat adjusted to -20°C. Once cut, sections were stored at -80°C until further use. For immunofluorescent staining, sections were air-dried, fixed in 50/50
Acetone/Methanol mix for 10 min at -20°C and air-dried. Sections were rehydrated in
PBS and non-specific binding was blocked using Dako protein-free block (Dako) for lh at room temperature in a humidified chamber. Sections were then incubated for lh in rat anti-CD31 antibody (1:50; Hyclone) followed by 30 min in donkey anti-rat FITC (1:200;
Jackson ImmunoResearch). After washing, sections were incubated in anti-phospho-
VEGFR2 primary antibody (1:100; Cell Signaling) or anti-VEGFR2 (1:400; Cell
Signaling) in PBS/0.3% Triton overnight at 4°C. The following day sections were
46 washed in PBS, followed by 30 minute incubation with goat anti-rabbit Cy3 secondary antibody (1:400; Jackson Laboratories). Slides were washed again in PBS and incubated for 2 minutes in nuclear stain DAPI, washed briefly in water and mounted using
Flourescent Mounting media (Dako).
Sections were examined by epifluorescent microscopy and images captured with a
20X objective in a blinded fashion. The total number of blood vessels and their VEGFR2 staining patterns were counted. Optimas 6.2 image analysis software program
(MediaCybernetics, San Diego, CA) was used to determine the total number of blood vessels for each field to obtain the blood vessel density (blood vessels per square millimeter).
Statistical Analysis
Calculation of preliminary summary statistics such as mean, standard deviation and standard error as well as graphing all the data was completed using Microsoft Excel
(Microsoft). On all samples, Grubbs' test, also called the ESD method (extreme studentized deviate), was used to determine significant outliers. Once outliers, if any, were identified and omitted from the analysis, ANOVA was perform to determine the significance within and between groups (p<0.05). Further, least significant difference
(Tukey) test was used to determine whether there were significant differences between groups. Data were presented as mean, standard error and range.
47 RESULTS
VEGF and VEGFR2 expression in primary and metastatic melanoma cell lines in
vitro
RT-PCR analysis revealed the presence of VEGFR2 mRNA in both primary and metastatic melanoma cell lines. Brain endothelial cells (bEND3) were used as a positive control while human fetal kidney cells (293T) were used as a negative control for
VEGFR2 mRNA expression (Figure 1 A).
Western blot analysis confirmed the expression of VEGFR2 protein in both primary and metastatic melanoma cell lines. Moreover, VEGFR2 expression was significantly higher (-20%) in WM115 primary than WM239 metastatic melanoma cells
(Figure IB). In both experimental protocols, BAECs were used as a positive control for
VEGFR2. WM115 melanoma cells express about 40-50% less VEGFR2 protein than
BAECs, while WM239 melanoma cells express about 80-90% less VEGFR2 protein than
BAECs (Figure IB).
Neutralization of VEGF-A ligand in vitro
We demonstrated through ELISA that the humanized ant i-VEGF antibody bevacizumab is able to neutralize VEGF ligand produced by our melanoma cell lines.
Bevacizumab (150 (ig/ml) significantly neutralized available VEGF in the conditioned media of primary melanoma cells by 24h but not at 6h, and significantly neutralized
VEGF produced by metastatic melanoma cells after both 6 and 24h at both 50 and 150
48 fig/ml (Figure 2). In addition, there was significant multi-fold increase in the production of VEGF ligand by metastatic compared to primary melanoma cells (*p<0.05).
Functional autocrine and intracrine VEGF/VEGFR2 signaling loops
To determine if both primary and metastatic melanoma cells have a functional autocrine VEGF/VEGFR2 signaling loop, western blot analysis was performed on pervanadate (ImM) treated whole cell lysates using two antibodies specific for detection of phosphorylated VEGFR2. We detected VEGFR2 phosphorylation at Tyr951 in both nuclear and cytosolic fractions, as well as total cell lysates (Figure 3A). In addition, the phosphorylation of VEGFR2 at this site is decreased in WM239 cells compared to
WM115 cells in both cytosolic and nuclear fractions. In contrast, levels of VEGFR2 phosphorylated at Tyrll75 were undetectable in separate nuclear and cytosolic (but not in the total cell lysates) of WM239 cells (Figure 3B). These results suggest the existence of both an autocrine and also an intracrine VEGF/VEGFR2 signaling loop in both primary and metastatic melanoma cells and the preferential phosphorylation of the
Tyr951 site over Tyrl 175 in downstream signaling. To confirm the localization of native and phosphorylated VEGFR2, we used fluorescently tagged antibodies against both phosphoVEGFR2 at Tyr951 and native VEGFR2 on cultured cells of primary and metastatic melanoma cell lines. These revealed the presence of phosphorylated and native
VEGFR2 in both plasma membrane and nucleus (Figure 4).
When WM115 cells were treated with either ZM323881 inhibitor (Whittles et al.,
2002) alone or in combination with bevacizumab, we confirmed that the VEGF/VEGFR2 autocrine signaling loop is functional in these cells. Bevacizumab failed to inhibit
49 VEGFR2 phosphorylation 15 and 30 minutes after its addition to the media (Figure 5).
Interestingly, we observed increased phosphorylation ofVEGFR2 receptor at Tyr951 and
Tyrll75 in both nuclear and cytosolic fractions when cells were treated with bevacizumab, compared to untreated controls (Figures 5 and 6). There was no detectable
VEGFR2 phosphorylation at Tyrl 175 in nuclear fractions. In addition, there is a decrease in phosphorylated VEGFR2 at Tyr 951 15 minutes after treatment with ZM323881 and
30 minutes after treatment with both ZM323881 and bevacizumab in cytosolic fractions.
In cytosolic fractions, downstream signaling was affected only at FAK: phosphorylated and native FAK decreased in ZM323881 and combination treatments at 15 minutes but returned to control levels at 30 minutes, suggesting VEGF signaling is mediated through
FAK in primary malignant melanoma cell line WM115. Activation of other downstream molecules such as Akt and p44/42 MAPK did not change in either of the treatment groups. However, prolonged exposure (3, 6 and 24 hours) to bevacizumab caused increased VEGFR2 phosphorylation at Tyr951 in primary melanoma cell line WM115
(Figure 7).
In addition, when WM239 cells were treated with the same agents as WM115 cells, in vitro (bevacizumab alone and in combination with ZM323881), we observed (in cytosolic fractions) downregulation of phosphorylated VEGFR2 at Tyr951 30 minutes after treatment with combination of bevacizumab and ZM323881 (Figure 8). There was no detectable phosphorylated VEGFR2 at Tyrl 175 in cytosolic fractions although
VEGFR2 native protein was readily detectable in these WM239 cell lysates. As observed in WM115 cell line, phosphorylation of VEGFR2 increased upon exposure to bevacizumab at both 15 and 30 minute exposures. Moreover, the amounts of
50 phosphorylated FAK also increased in all treatment groups compared to controls. Levels of phosphorylated and native Akt did not change from control levels. We also detected native VEGFR2 in nuclear fractions of WM239 protein lysates, while phosphorylated
VEGFR2 was only detected at Tyr951 and not at Tyrll75, as previously observed with cytosolic fractions (Figure 9). Treatments with either ZM323881 or bevacizumab do not appear to change the levels of native or phosphorylated VEGFR2 at Tyr951 in nuclear fractions.
Bevacizumab is antiangiogenic in primary and metastatic melanoma xenografts
In order to test the functionality of this VEGF/VEGFR2 autocrine signaling loop and test the therapeutic properties of bevacizumab in melanomas, we treated mice bearing WM115 or WM239 xenografts. We found no significant differences in average tumor volume in mice treated with bevacizumab compared to control for both primary melanoma (WM115) and metastatic melanoma (WM239) (Figure 10A and 10B).
However, we observed significant differences (*p<0.05) in blood vessel density between bevacizumab and control treated mice with both melanoma xenografts (Figure
11 A). We also evaluated VEGFR2 receptor staining in blood vessels, but no significant differences were observed between bevacizumab and control treated mice with either primary or metastatic melanoma (Figure 1 IB). Interestingly, approximately 10% of the blood vessels in all groups were negative for VEGFR2 expression - pointing towards the existence of heterogeneous VEGFR2 expression in blood vessels of melanomas.
In addition, overall VEGFR2 expression was evaluated in total tumor lysates, revealing no significant (p>0.05) differences between treatment groups. Although there
51 was a trend towards reduced VEGFR2 levels in treated tumors, this was not significant
(p>0.05) (Figure 12A and 12B).
Moreover, when we investigated the levels of VEGF in WM115 and WM239 xenografts treated with bevacizumab, we observed that bevacizumab treated WM239 tumors had significantly less detectable VEGF (*p<0.05) than control xenografts.
WM115 xenografts did not show this difference (p>0.05) (Figure 12C).
Downregulation of VEGFR2 in severe hypoxia
Western blot analysis showed that hypoxia significantly (*p<0.05) decreased the expression of VEGFR2 receptor in WM115 primary and WM239 metastatic melanoma cells at both 24 and 48h (normalized to a -tubulin). In fact, hypoxia almost completely diminished WM239 expression of VEGFR2 (Figure 13A and 13B).
52 DISCUSSION
Although the action of VEGF in the process of tumor angiogenesis via VEGFR signaling by tumor endothelium is well known, its role via VEGFR signaling of cancer cells is less clear. The expression of endothelial receptors, such as VEGFR2, on cancer cells that are not of endothelial origin has been well documented (Bellamy et al., 1999;
Boocock et al., 1995; de Jong et al., 1998; Decaussin et al., 1999; Dias et al., 2001; Ferrer et al., 1999; Graells et al., 2004; Graeven et al., 1999; Lazar-Molnar et al., 2000; Liu et al., 1995; Sher et al., 2009). Although the functions of VEGFR family members on cancer cells are not completely understood, the concomitant expression of VEGF and
VEGFR suggests that these receptors might mediate biological effects in an autocrine fashion (Lazar-Molnar et al., 2000). Most of the studies suggested a survival advantage since VEGF is generally produced in large quantities by most cancer cells (Graells et al.,
2004). Binding of VEGF to its corresponding receptor, VEGFR2 would give cancer cells an opportunity to maintain their own survival without dependence on other factors.
Presence of active autocrine VEGF/VEGFR2 loop in breast cancer has been associated with increased activation of p38 MAPK (Aesoy et al., 2008). Dias et al. showed that functional VEGF/VEGFR2 autocrine loop in leukemia supports leukemia cell survival and migration (Dias et al., 2001). Functional autocrine VEGF/VEGFR2 loop has also been identified in head and neck carcinoma cells (Neuehrist et al., 2001). In bladder cancer, VEGFR2 positive bladder cancer cell lines demonstrated increased invasion in vitro (Xia et al., 2006). In melanomas, as in endothelial cells, VEGFR2 is major mediator
53 of the mitogenic, angiogenic and permeability-enhancing effects of VEGF (Zachary and
Gliki, 2001) and is not normally expressed by melanocytes (Gitay-Goren et al., 1993).
Our data confirm the presence of a functional autrocrine VEGF/VEGFR2 signaling loop in human malignant melanoma cell lines WM115 and WM239. When we quantified expression levels of VEGFR2 in cells derived from both primary and metastatic lesions, significantly lower VEGFR2 expression was seen with metastatic derived cells (WM239) compared to WM115 as well as to normal endothelial cells. The reasons behind this may be due to the malignant phenotype. In fact, its been shown that aggressive melanoma tumor cells may "masquerade" as other cell types and express genes associated with many other cellular phenotypes such as endothelial, epithelial, pericyte, fibroblast and haematopoietic and cells (Bittner et al., 2000; Seftor et al., 2002).
These indicate that aggressive melanoma cells may revert to an undifferentiated, embryonic-like phenotype, although the reasons behind this are still to be elucidated
(Seftor et al., 2002). Melanomas have been reported capable of "vascular mimicry" - expression of endothelium-associated genes and formation of vascular networks similar to formation of embryonic vascular networks (Folberg et al., 2000; Folberg and Maniotis,
2004; Maniotis et al., 1999). In addition, as previously mentioned, other studies reported that concomitant expression of VEGF and VEGFR2 on melanoma cells promotes their growth and survival (Graells et al., 2004).
Cells from metastatic melanoma significantly up-regulated their VEGF expression compared to cells from primary melanoma. This is not surprising since the ability of melanomas to produce large amounts of VEGF is one of their hallmarks (Graells et al.,
2004; Graeven et al., 1999; Salven et al., 1997) and hyperpermeability of the newly
54 formed blood vessels, caused by VEGF, plays an important role in melanoma metastasis
(Salven et al., 1997). The overproduction of VEGF by these cell lines compared to normal endothelial cells may trigger VEGFR2 receptor downregulation and degradation; therefore accounting for the reduced protein levels seen in metastatic cells compared to cells from primary lesions. Besides VEGFR2 (Dougher and Terman, 1999), which was shown to be internalized upon VEGF binding (Duval et al., 2003), several receptor tyrosine kinases, such as epidermal growth factor receptor (Ettenberg et al., 1999), platelet-derived growth factor receptor (Miyake et al., 1998) and colony-stimulating factor-l(Kallies et al., 2002) are actively degraded following the exposure to their respective ligands (Schlessinger, 2000; Thien and Langdon, 2001). This degradation is initiated by addition of ubiquitin moieties following receptor-ligand binding, which targets them to the lysosomal/proteasomal machinery for degradation.
In addition, our studies confirm that the anti-VEGF antibody, bevacizumab, is effective in neutralizing virtually all VEGF secreted by these melanoma cells. In spite of that, not only did bevacizumab fail to inhibit VEGFR2 phosphorylation in melanoma cells in vitro, it actually produced increased receptor activation that was sustained for several hours. This phenomenon has not been reported in the literature; in fact studies have previously shown that bevacizumab inhibits VEGFR2 phosphorylation in tumor vessels of breast cancer (Wedam et al., 2006), lung cancer (Inoue et al., 2007) and human microvascular endothelial cells (Costa et al., 2009). It is only speculation at this point but bevacizumab may have somehow bound to the receptor in our model and activated it.
More studies need to be done to determine why this occurred.
55 In vitro studies with primary melanoma cell line using the VEGFR2 small molecule inhibitor ZM323881 revealed that inhibition by ZM323881 only affected phosphorylated and native FAK, involved in cellular adhesion and migration, but none of the other downstream signaling molecules tested, pointing towards the functionality of this VEGF signaling pathway in melanoma. In fact, loss of detectable phosphorylated and native FAK points to the rapid degradation or possible translocation of this molecule, the mechanisms of which still need to be investigated in future studies. Translocation of phosphorylated FAK to the nucleus has been reported before, suggesting this as a regulatory mechanism during apoptosis of human umbilical vein endothelial cells (Lobo and Zachary, 2000). Generally, FAK mediates many cellular processes, such as survival, migration and invasion (Hanks et al., 2003). High levels of FAK are expressed in metastatic cancer cells, although the precise role of FAK in metastasis is not completely understood (Baish and Jain, 2000). In fact, up-regulation of FAK protein expression and uncontrolled signaling through it were held responsible for promotion of malignant phenotype, resistance to chemotherapy and radiotherapy in melanoma. Preclinical studies have shown that inhibition of FAK signaling causes inhibition of metastasis in melanoma
(Goundiam et al., 2009; Kaneda et al., 2008; Yang et al., 2009) as well as melanoma cell apoptosis (Sun et al., 2009). Hence, FAK is currently being evaluated as an antiangiogenic therapy target candidate (Chatzizacharias et al., 2007).
A study by Lacal et al. showed that human melanoma cells that simultaneously produce VEGF-A and express VEGFRs exhibit a higher spontaneous ability to invade the extracellular matrix (ECM) than melanoma cells not expressing either VEGF-A or
VEGFRs (Lacal et al., 2005). When the same treatments were administered with WM239
56 cells, derived from metastatic melanoma, we did not observe any significant changes in the phosphorylation of VEGFR2. Again, phosphorylation levels seem to increase during bevacizumab treatment and no detectable phosphorylation at the Tyrl 175 was observed, pointing to the fact that Tyr951 is major phosphorylation site for these cells and that it probably plays an important role in migration and invasion of melanoma, as previously suggested.
Although bevacizumab caused a decrease in the available VEGF in WM239 tumor xenografts, this decrease did not inhibit growth of either primary or metastatic melanoma xenografts in vivo suggesting a complex mechanism of regulation of VEGF and VEGFR2 in melanomas. Bevacizumab was antiangiogenic in both primary and metastatic melanoma, which did not result in decreased tumor volume in either of the melanoma xenografted models. This could be due to our observed increased signaling through VEGFR2 in melanoma cells treated with bevacizumab described above.
Besides an autocrine VEGF/VEGFR2 signaling loop, we also documented the presence of intracrine VEGF/VEGFR2 signaling which allows cancer cells to stimulate their own survival pathways without the need for exogenous secreted factors. In other similar studies, autocrine and intracrine VEGF/VEGFR2 signaling loops regulated survival of subsets of acute leukemia cells (Dias et al., 2001; Zhang et al., 2004). Lee et al. documented a unique survival system in breast cancer cells by which VEGF acted as an intracrine survival factor through its binding to VEGFR1 (Lee et al., 2007). Although growth factors such as VEGF are generally seen as being operative at the cell surface, there is also evidence that VEGF can be translocated to the nucleus (Li and Keller, 2000) at the sites of wound healing. In fact some other growth factors such as PDGF (Lee et al.,
57 1987), can also translocate to the nucleus via nuclear localization signals. Besides growth factors, some receptors such as EGFR are used in clinical trials as targets for cancer treatment (Dittmann et al., 2010). VEGFR2 can also be internalized and remains in endosomal compartments for longer periods of time where it continues to be phosphorylated and activate p44/42 MAPK phosphorylation and cell proliferation.
Internalization of VEGFR2 in this case occurred as a consequence of contact inhibition in confluent endothelial cell monolayers that as such respond poorly to VEGF (Lampugnani et al., 2006).
Similar intracellular signaling presumably occurs in intracrine pathways, where ligand/receptor interactions are completely intracellular. Such internal signaling systems imply that anti-VEGF neutralizing agents used in clinical trials today (such as bevascizumab and aflibercept) may not effectively block those pathways inside cancer cells. Since cancer cell expression of VEGFRs is more prevalent that originally thought, this could be a significant barrier to effective antiangiogenic therapy in many types of tumors. In our studies, we show evidence of both autocrine as well as intracrine
VEGF/VEGFR2 signaling loops in malignant melanoma. VEGF signaling in our cells could be inhibited, in most instances, by use of small-molecule inhibitors such as
ZM323881 that readily penetrate the cell surface (Whittles et al., 2002). Moreover, nuclear fractions revealed phosphorylation of VEGFR2 at Y951 while phosphorylation at
Y1175 was non-detectable in our system, suggesting the importance of this pathway in melanoma cells. In ligand-induced phosphorylation of VEGFR2 only Y1175 auto- phosphorylation leads to VEGF-dependent endothelial cell proliferation (Matsumoto and
Mugishima, 2006; Takahashi et al., 2001) while endothelial cell migration towards a
58 gradient of VEGF is due to the phosphorylation of Y951 residue on VEGFR2, which then recruits the adapter molecule TSAd resulting in stimulation of the migration signal
(Matsumoto et al., 2005; Zeng et al., 2001b).
We also investigated the effect of severe, prolonged (24 and 48h) hypoxia on primary and metastatic melanoma cell VEGFR2 expression and found that this receptor is significantly downregulated in hypoxia to almost undetectable levels in metastatic melanoma cells. This downregulation is probably due to the over-expression of VEGF in hypoxic conditions since VEGF synthesis is regulated by hypoxia (Detrnar et al., 1997).
As discussed above, this over-expression of VEGF could cause over-stimulation and eventual internalization and degradation of VEGFR2 by melanoma cells. This renders anti-VEGF therapies, such as bevacizumab, potentially refractory for the areas of tumors that are hypoxic and may be another reason for mixed success observed with bevacizumab in clinical trials. Receptor tyrosine kinase small molecule inhibitors, such as
ZM323881, may be more effective in treatments of hypoxic cells than monoclonal antibodies due to their ability to penetrate cells easier and thus treat tumors with functional intracellular autocrine VEGF signaling (Gerber et al., 2002; Lee et al., 2007).
Taken together our findings demonstrate that both primary and metastatic melanoma cells may have functional autocrine and intracrine VEGF/VEGFR2 signaling loops, which may aid in survival of cancer cells, via signaling through PI3K and Akt. In fact, we report melanoma cells in our model preferentially show autophosphorylation at
Tyr951, a site that signals migration, over Tyrl 175, a site that signals survival. We also found that bevacizumab is an effective antiangiogenic but not anti-tumorigenic agent in human melanoma xenografted to mice and that it may have undesirable effects on
59 melanoma cells by increasing the phosphorylation of VEGFR2 for prolonged periods.
These results suggest that in case of cancers with intracrine VEGF/VEGFR2 signaling loops, small-molecule inhibitors of VEGFR2 may be more effective than neutralizing antibodies at disease control.
60 fN 1.2 cd IX 1 a w I 0.8 > m >
BAEC WM115 WM239
Figure 1. Expression of VEGFR2 in primary and metastatic melanoma cells in vitro (A) An image of agarose gel stained with ethidium bromide showing VEGFR2 cDNA amplicons in both primary (WM115) and metastatic (WM239) melanoma cell lines. (B) A representative western blot of VEGFR2 protein expressed by BAECs (positive control), WM115 and WM239 cells, a-tubulin was used for normalization purposes. Densitometry of these blots shows significant differences in the expression of VEGFR2 in melanoma cell lines compared to each other (**) as well as control (*) (*p < 0.05, **p<0.05) N = 3.
61 • WM115 6h • WM239 6h • WM115 24h • WM239 24h
ES£±L
Control Bevacizumab Bevacizumab (50 ng/ml) (150 (ig/ml)
Figure 2. Effects of Bevacizumab on the VEGF expression of primary and metastatic melanoma in vitro. Results of VEGF ELISA from serum free conditioned medium shows that metastatic melanoma cells significantly up- regulated VEGF expression compared to primary melanoma cells. In addition, significantly higher VEGF expression was detectable in controls than in WM115 and WM239 cell lines treated with bevacizumab (50 or 150 ng/ml) (*p<0.05)N = 3.
62 Total Cytosolic Nuclear pVEGFR2 230 kDa Tyr951 230 kDa- VEGFR2
a-tubulin/ 55 kDa lamin
B Total Cytosolic Nuclear pVEGFR2 230 kDa Tyrl 175
230 kDa- VEGFR2
a-tubulin/ 55 kDa- lamin
— J3 —U-) OSm ON — £2 — Figure 3. Detection of phosphorylated VEGFR2 receptor in primary and metastatic melanoma lysates. Representative western blots show native and phosphorylated (A) at Tyr951 (B) at Tyrl 175 VEGFR2 and in both in primary and metastatic melanoma cells in total, nuclear and cytosolic protein lysates. Lamin or a-tubulin was used for normalization purposes. VEGFR2 phosphorylation at Tyrl 175 is only seen in total protein cell lysates. Presence of phosphorylated VEGFR2 in both cytosolic and nuclear fractions suggests the possibility of active autocrine and intracrine signaling. 63 I VEGFR2 DAPI HyEGFR2 Neg. ^ DAPI I I pVEGFR2 lpVEGFR2(Tyr951) DAPI (Tyr951) DAPI (N Figure 4. Detection of phosphorylated VEGFR2 receptor in primary and metastatic cultured melanoma cells. Immunofluorescent staining showing native and phosphorylated VEGFR2 in primary and metastatic melanoma cells. There was no detectable staining in negative controls. Note the presence of both native and phosphorylated VEGFR2 in both cytoplasm (arrows) and nucleus (asterisks) of melanoma cells. Scale bar is 10 jum. 64 rV^r0 15 30 ^mi n Bevacizumab --+ - + - + - + ZM323881 + + + + pVEGFR2\ 230 KDa Tyr951 \ pVEGFR2 230 KDa Tyrl 175 230 KDa » VEGFR2 pFAK 120 KDa IHIIII Cytosolic 120 KDa uwi > Fraction 60 KDa pAkt Ser473 pan Akt 60 KDa 55 KDa a-tubulin 44 KDa p-p44/42J Figure 5. Inhibition of VEGFR2 phosphorylation in primary melanoma cell cytosolic fractions in vitro. Representative western blots of cytosolic fractions of WM115 show inhibition of VEGFR2 phosphorylation at Tyr951 at 15 minutes in ZM323881 treated samples and at 30 minutes in combination samples. Phosphorylation at Tyrl 175 decreases at 15 and 30 minutes in combination treatment. Interestingly, treatment with bevacizumab alone results in increased phosphorylation at both 15 and 30 minutes for both Tyr951 and Tyrl 175 compared to controls. In addition, ZM323881 alone and in combination with bevacizumab caused decreased phosphorylation of FAK and almost complete downregulation of native protein at 15 minutes. There were no significant changes in the levels of pAkt, pan Akt, tubulin, or phospho- p44/42 MAPK in cytosolic proteins. 65 0 15 30 min Bevacizumab - - +- + - +- + ZM323881 - + + + + pVEGFR2 230 KDa . P-'IIB Tyr951 pVEGFR2 230 KDa » Nuclear Tyrl 175 f Fraction 230 KDa VEGFR2 72 KDa lamin Figure 6. Determination of the inhibition of VEGFR2 phosphorylation in primary melanoma cells nuclear fractions in vitro. In nuclear fractions, there was decrease in phosphorylation of VEGFR2 at Tyr951 in ZM323881 treated cells at 15 and 30 minutes, and decreased phosphorylation in the combination treated cells at 30 minutes, compared to controls. In addition, there was increased phosphorylation of VEGFR2 in bevacizumab treated cells at both 15 and 30 minutes. There was no detectable phosphorylation of VEGFR2 at Tyrl 175. Lamin was used for normalization purposes. 66 6 24 hours pVEGFR2 230 KDa Tyr951 230 KDa VEGFR2 55 KDa a-tubulin Bevacizumab + + + Figure 7. Long-term inhibition of VEGFR2 phosphorylation in primary melanoma cells in vitro. Composite western blots showing that exposure of WM115 cells to bevacizumab for extended periods of time (1,6 and 24 hours) produced increase in phosphorylation of VEGFR2 at Tyr951 compared to controls, a-tubulin was used for normalization. 67 15 30 mm A. rS—K V Bevacizumab + - + -+- + - + + - - + + ZM323881 pVEGFR5\ 230 KDa Tyr951 230 KDa Cytosolic pAkt Fraction 60 KDa Ser473 pan Akt > 60 KDa pVEGFR2 230 KDa Tyrl 175 230 KDa I < VEGFR2 55 KDa a-tubulinJ Figure 8. Inhibition of VEGFR2 phosphorylation in metastatic melanoma cells in vitro. Representative western blots of cytosolic fractions of WM239 show that when WM239 cells were treated with bevacizumab in combination with ZM323881, we observed downregulation of phosphorylated VEGFR2 at Tyr951 after 30 minutes of treatment. There is no detectable phospho- VEGFR2 at Tyrl 175 in cytosolic fractions but VEGFR2 native protein was readily detectable in these cytosolic fractions of WM239 cell lysates. Phosphorylation of VEGFR2 and FAK increased with bevacizumab treatment after both 15 and 30 minutes of treatment. Levels of phosphorylated and native Akt did not change. 68 0 15 30 min rS—K V \ Bevacizumab . + . + . + . + ZM323881 . - + + - - + + A 230 KDa pVEGFR2 Tyrl 175 230 KDa VEGFR2 V Nuclear mmrnmmmmmm r Frac,,on 230 KDa VEGFR2 72 KDa lamin J Figure 9. Inhibition of VEGFR2 phosphorylation in metastatic melanoma cells nuclear fractions in vitro We also detected native VEGFR2 in nuclear fractions of WM239 protein lysates, while phosphorylated VEGFR2 was only detected at Tyr951 and not at Tyrl 175, as previously observed with cytosolic fractions. Treatments with either ZM323881 or bevacizumab do not change the levels of native or phosphorylated VEGFR2 at Tyr951 in WM239 cell nuclear lyates. 69 • Control • Bevacizumab | 300 £ 200 1 100 3 H 0 0 5 10 15 20 25 30 Treatment day B 300 •Control I 250 ^ 200 • Bevacizumab s I 150 > 100 E=S- 50 1 —i— 10 20 30 40 Treatment Day Figure 10. Impact of bevacizumab on growth of primary and metastatic melanoma cell xenografts. Tumor volume in WM115 (A) and WM239 (B) mouse xenografts did not differ significantly between control and bevacizumab groups at any point during the treatment (p>0.05) N=8. 70 M •WM115 C 90 I 80 ° WM239 >.70 "K 60 50 13 40 % 30 § 20 S 10 ii o Control Bevacizumab B * * H' * i * VEGFR2 CD31 overlay W 80 ^ 75 Control Bevacizumab Figure 11. Impact of bevacizumab on angiogenesis of primary and metastatic melanoma cell xenografts (A) Significant differences (*p<0.05) in average blood vessel density between control and bevacizumab treated groups in both primary and metastatic melanoma xenografts, N = 8. (B) Immunofluorescence was used to determine that approximately 90% of blood vessels in melanoma xenografts are VEGFR2 positive (star) while approximately 10% did not show detectable VEGFR2 expression (arrow). There were no significant differences in the percentage of VEGFR2 positive or negative blood vessels between control and bevacizumab treated groups (p > 0.05) N = 8. Scale bar is 30 jum 71 WM115 WM239 Control Bevacizumab Control Bevacizumab 230 kDa - VEGFR2 55 kDa- a-tubulin B s "S VI t 2.5 a x a> WM115 2 [—1 WM239 U. 1.5 O W > 1 •B 0.5 •h-F 06 0 Control Bevacizumab 300.0 2*250.0 •WM115 is Fx|WM239 S 200.0 a fc 150.0 a w 100.0 50.0 0.0 Control Bevacizumab Figure 12. Expression of VEGFR2 in melanoma xenografts treated with and without bevacizumab. (A) Representative western blots of lysed WM115 and WM239 xenografts show (B) no significant differences (p>0.05) between control and bevacizumab treated groups in expression of VEGFR2 receptor in vivo N=4. (C) When VEGF was quantified using ELISA in lysed tumor xenografts of WM115 and WM239 cells, significant differences (*p<0.05) were observed in the expression of VEGF in bevacizumab treated group compared to control. N=3 72 WM115 WM239 24h 48h 24h 48h 230kDa — * VEGFR2 55 k£)a ^ O-tubulill hypoxia . + .+ .+ . + B 1 1,8 °24h 4S>" 1,6 48h e 1 4 1 •M 1 a j § 0,8 W 0,6 « 0,4 * 2 I °> ±2 IS 0 control hypoxia Figure 13. Expression of VEGFR2 receptor in primary and metastatic melanoma cell lines exposed to hypoxic conditions in vitro. (A) Representative, composite blots showing expression of VEGFR2 receptor in primary (WM115) and metastatic (WM239) melanoma cell lines when exposed 24 and 48 hours of hypoxia, a-tubulin was used for normalization purposes. (B) Quantification demonstrates significant decrease in VEGFR2 expression in primary (WM115) melanoma cells when exposed to hypoxia for 24 and 48 hours ( p< 0.05), N = 3. 73 CHAPTER 3 - Antiangiogenic activity of metronomic cyclophosphamide in human melanoma xenografts is modulated by Tie2 inhibition 74 INTRODUCTION Studies have shown that chemotherapeutic drugs may have antiangiogenic properties when administered metronomically at doses significantly lower than maximum tolerate dose (MTD) and that as such appear to have much less severe or even absent cytotoxic side effects (Emmenegger et al, 2004). Cyclophosphamide (CTX), a nitrogen mustard alkylating agent (Coggins et al., 1960; Foye et al., 1960), is clinically the most studied drug in a low dose metronomic chemotherapy setting (Colleoni et al., 2006; Garber, 2002). Currently, it is used clinically at MTD with other chemotherapy agents in the treatment of lymphoma (Michallet and Coiffier, 2009), leukemia (Smolej, 2009), ovarian cancer (Reed et al., 1995; Thigpen et al., 1993) and some other solid tumors. In contrast to MTD, metronomic administration of low dose cyclophosphamide does not kill cancer cells but instead induces selective apoptosis of genetically stable endothelial cells (hence circumventing drug resistance) in tumors (Browder et al., 2000). In addition, studies by Hamano et al. suggest that this effect is due to the overproduction of Thrombospondin-1 (TSP-1), a well known, highly specific and potent endogenous inhibitor of angiogenesis (Bocci et al., 2003; Dawson et al., 1997; Hamano et al., 2004; Lawler, 2002; Nor et al., 2000; Rodriguez-Manzaneque et al., 2001). In addition, antiangiogenic effects of metronomic low dose CTX are lost in TSP-1 knockout mice compared to wild type control (Bocci et al., 2003; Hamano et al., 2004). TSP-1 has anti- proliferative and pro-apoptotic effects on endothelial cells, primarily by binding to the CD36 membrane receptor (Dawson et al., 1997). Interestingly, an inverse relationship between TSP-1 and VEGF production has been reported in cultured ovarian epithelial 75 cells (Greenaway et al., 2007) and low dose metronomic cyclophosphamide (LDM CTX) has been shown to decrease VEGF levels in patients with breast cancer (de Jong et al., 1998), suggesting a possible relationship between VEGF expression and LDM CTX induced TSP-1 expression. In general, the ant i-tumor effects of cancers treated with LDM chemotherapy are even more pronounced when combined with an antiangiogenic inhibitor that targets endothelial cells specifically (Gasparini, 2001). Tie2 receptors are primarily found on endothelial cells and are constituently expressed in normal vasculature (Wong et al., 1997). Thus, it was surprising when our laboratory identified blood vessels within human cancers that lacked Tie2 expression. In particular, malignant melanoma had the highest percentage (-15%) of Tie2 negative blood vessels of all cancer types evaluated (Fathers et al., 2005). In addition, primary melanoma xenografts treated with a Tie2 inhibitor (TekdeltaFc) showed no response while colorectal cancer xenografts (with lowest percentage of Tie2 negative blood vessels) showed significant inhibition of tumor growth, supporting the concept that vascular phenotype impacts response to targeted antiangiogenic therapy (Fathers et al., 2005). Differential expression of vascular receptors, such as Tie2, may be due to tumor microenvironment conditions such as hypoxia and hypoglycemia. Hypoxia occurs as a result of an imbalance between the oxygen supply and consumption (Hockel and Vaupel, 2001a; Hockel and Vaupel, 2001b). Evidence suggests that 50-60% of locally advanced solid tumors (Lartigau et al., 1997; Vaupel et al., 2007; Vaupel et al., 2001) may exhibit hypoxic areas of tissue heterogeneously distributed within the tumor mass. The pC>2 measurement values in patients with melanoma were 11.6 mmHg (or 1.6% O2) for 76 primary tumors, 17.1 mmHg (or 2.4% O2) in the affected lymph nodes and 6.7 mmHg (0.9% O2) in skin metastases, compared to the median pC>2 of 40.5 mmHg (or 5.7% O2) for normal tissues (Lartigau et al., 1997; Vaupel et al., 2001). In fact, more than half of all human clinical specimens examined had 0-2.5 mmHg of pC>2 in their tumor mass. Stringent hypoxia/anoxia (<0.5% O2) can promote tumor progression by selecting cells with mutations that allow them to survive in these extreme conditions (Graeber et al., 1996; Hockel and Vaupel, 2001a; Vaupel and Mayer, 2005), with low responsiveness to radiation and chemotherapy (Hockel and Vaupel, 2001a). Invasive cancers also have an abnormal energy metabolism, which, under persistent hypoxia, leads to constitutive upregulation of glycolysis (Gatenby and Gillies, 2004). The consequences of increased glycolysis require further adaptation to environments with high acid and low glucose concentrations (Gatenby and Gillies, 2004). A common property of invasive cancers is an abnormal energy metabolism characterized by high aerobic glycolysis (Gogvadze et al., 2009; Sattler et al., 2009; Vander Heiden et al., 2009; Warburg, 1961; Warburg et al., 1965). Exertion of selective pressures, such as persistent hypoxia, leads to constitutive upregulation of glycolysis as a result of mutations or epigenetic changes, even in the presence of oxygen. The consequences of increased glycolysis require further adaptation to environments with high acid and low glucose concentrations (Gatenby and Gillies, 2004). In the process of glycolysis, glucose is converted to pyruvate and then to lactic acid, that can be utilized for energy purposes by other tissues or transported to the liver for resynthesis of glucose (Cori cycle) (Waterhouse, 1974a; Waterhouse, 1974b). Although tumor energy metabolism has not been intensely researched, widespread clinical use of 18- 77 fluorodeoxyglucose (FdG) positron-emission tomography (PET) has demonstrated that the glycolytic phenotype is indeed observed in most human cancers, and that it may be an important clinical target. Consistent with the FdG PET results, the glycolytic rate in cultured cell lines seems to correlate with tumor aggressiveness (Barwick et al, 2009; Ford et al., 2009; Jadvar et al., 2009). In fact, cells derived from tumors often maintain their metabolic phenotypes in culture under normoxic conditions, indicating that aerobic glycolysis is constitutively upregulated through stable genetic or epigenetic changes (Gatenby and Gillies, 2004). To date, there are no studies that examined the effect of hypoglycemia on Tie2 expression. Heterogeneous expression of Tie2 in tumor vasculature suggested a role for Tie2 in tumor angiogenesis, however, elucidating the functional significance of Tie2 expression in tumors requires the use of a specific Tie2 inhibitor, TekdeltaFc, which is an artificial extracellular domain of Tie2 (Das et al., 2003). Since angiopoietins bind with high affinity to Tie2 extracellular domain, we reasoned that this inhibitor should efficiently interfere with angiopoietin-mediated Tie2 activity. In addition, we hypothesized that the tumor microenvironment is at least partially responsible for the lack of Tie2 expression observed in malignant melanoma blood vessels, and investigated the possibility that Tie2 heterogeneity is due to severe hypoxia, hypoglycemia and/or tumor secreted factors. Numerous recent publications suggest a clear advantage of simultaneously using multiple antiangiogenic therapies in combination with metronomic, low dose chemotherapy, thus targeting more than one endothelial cell signaling pathway (Brown et al.; Daenen et al., 2009; Dellapasqua et al., 2008). Therefore, we examined the impact of 78 combined antiangiogenic approaches on endothelial Tie2 signal transduction pathways, metastasis-derived malignant melanoma cancer cell growth as xenografts, and tumor blood vessel phenotype. We wanted to determine if the response to targeted antiangiogenic therapy such as Tie2 inhibition can be enhanced by non-targeted antiangiogenic therapy such as LDM CTX. 79 MATERIAL AND METHODS A list of all chemicals and suppliers as well as the description of materials and solutions prepared for the following experiments are detailed in Appendices I and II, respectively. Cell lines and reagents Human melanoma cell line WM115, isolated from a primary lesion (Rodeck et al., 1987) and WM239 isolated from the same patient's secondary lesion (Rodeck et al., 1987), human colorectal cancer cell line HCT116 (Crowley-Weber et al., 2002; Ishizu et al., 2007) and human fetal kidney cell line 293T (Graham et al., 1977) were purchased from ATCC (Manassas, VA, USA), while primary bovine aortic endothelial cells (BAEC) were isolated from aortae of adult cattle and characterized as previously reported (Coomber, 1995). All cells were maintained in Dulbecco's modified Eagle's Medium (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen), sodium pyruvate (Invitrogen) and gentamicin (Invitrogen) in a humidified atmosphere at 37°C in 5% C02 and 95% air (standard culture conditions). Tie2 inhibitor, murine TekdeltaFc was provided by Amgen. The metabolically active form of cyclophosphamide, 4-hydroxycyclophosphamide (4- HC), was purchased from NIOMECH, IIT GmbH. Treatment using TekdeltaFc and 4HC in vitro For studying Tie2 signaling pathway upon treatment with TekdeltaFc with and without 4-HC, BAECs cells were seeded and grown to confluence in standard culture conditions overnight, then treated with 5 jig/ml Tie2 inhibitor TekdeltaFc with and 80 without 100 ng/ml of 4-HC for 15, 30 and 60 minutes and 24 hours. Cells were treated with sodium pervanadate (1M) for 30 minutes before lysis, and total protein was collected using cell lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta- glycerophosphate, 1 mM Na3V04, 1 ng/ml leupeptin (Cell Signaling Technology) supplemented with 1 mM PMSF and 2 ng/ml aprotinine and stored at -80°C until further use. Similarly, for survival studies, BAECs were grown to confluence as previously described and treated alone and in combination with TekdeltaFc and 4-HC. Twenty-four, 48 and 72h post treatment, viable cells were trypsinized and counted using a haemocytometer and 4% trypan blue solution (Sigma-Aldrich) to differentiate between viable/dead cells. For tube-like formation on Matrigel (BD Biosciences), BAECs were plated at 10,000 cells per 24 well tissue culture plate, into wells pre-coated with 100 fil of Matrigel. Cells were then allowed to attach overnight in the presence or absence of TekdeltaFc (0, 10, 20 and 50 (ig/ml) and tube formation evaluated after 24 and 48 hr incubation. Additional cultures were treated 24h post plating with these reagents, and tube degeneration examined after an additional 24 and 48h Hrs. Exposure of endothelial cells to hypoxic, hypoglycemic and cancer cell conditioned medium BAEC were maintained in standard culture conditions, grown to confluence, serum-starved overnight and then deprived of oxygen, glucose or both oxygen and glucose. BAECs used as control were maintained in serum free DMEM under standard 81 culture conditions. Hypoxia/oxygen deprivation was simulated by placing BAECs in a Modular Incubator Chamber (Billups-Rothenberg Inc., Del Mar, CA) containing a humidified mixture of 95% N2 and 5% C02 (maintaining 02 content at <0.1%). BAECs were exposed to hypoxic conditions for 6, 12 and 24h. Hypoglycemia/glucose deprivation was simulated by growing cells in glucose-free media (Invitrogen) for 6, 12 and 24h; an additional set of BAEC plates were both oxygen and glucose deprived. Total cell lysates were collected at specified time points using lysis buffer (Cell Signaling Technology) supplemented with 1 mM PMSF and 2 (ig/ml aprotinine and stored at -80°C until further use for western blotting. BAECs were also exposed to serum-free conditioned media collected from WM239 and HCT116 cell lines after 48 hrs of exposure. After conditioned media collections, cells were trypsinized and counted for normalization purposes. Conditioned media was standardized for number of cells/ml of media. BAECs were grown to about 70% confluence in standard culture conditions. Next, cells were switched to 0.5% FBS/DMEM and incubated overnight. The following day, BAECs were treated for 24 hrs with various amounts of conditioned media equivalent to 0, 50,000, 15,0000, 300,000 and 500,000 cells/ml. Control wells were treated with 10% FBS/DMEM for 24 hrs. All cells were lysed as previously described and western blotting was performed. Western blotting Western blot was performed by running 10-20 jig total protein from cell lysates, or 25-45 fig of concentrated conditioned media (for TSP-1 experiments) on 7.5% SDS- PAGE gels and transferring proteins to PVDF membranes (Roche). Membranes were 82 blocked using 5% BSA (Jackson ImmunoResearch) in TBS/T for phosphorylated protein detection and 5% milk in TBS/T for all other protein detection, and incubated overnight in primary antibody diluted in blocking solution. Primary antibodies used were rabbit anti-phospho-Tie2 (Tyr992), rabbit anti-phospho-Akt (Ser473), mouse anti-Tie2, rabbit pan-Akt (all 1:1000; Cell Signaling Technology) and mouse anti-thrombospondin 1 (NeoMarkers). Mouse anti-a-tubulin (1:200,000; Sigma -Aldrich) was used for normalization purposes of cell lysates, and Amido black membrane staining was used to confirm equal loading of conditioned medium blots. Membranes were then washed and incubated in goat anti-rabbit or goat anti-mouse POD secondary antibody (1:10,000 dilution; Sigma-Aldrich) for 30 minutes, washed again, incubated with chemilluminescent reagent (Roche) and exposed to X-ray film (Konica). Once imaged, PVDF membranes probed for phospho-proteins were stripped in diluted Reprobe Plus stripping solution (Milipore) and re-probed for corresponding native proteins mentioned above. Growth of tumor xenografts All procedures described below were done according to the guidelines and recommendations of the Canadian Council of Animal Care and approved by the University of Guelph Local Animal Care Committee. Tumor xenografts were established in Tie2IacZ+/RAG1" mice (Fathers et al., 2005) by injecting 100 of 0.1% BSA/PBS solution containing lxlO6 WM239 melanoma cells subcutaneously into the right flank. Tumor growth was measured every few days using calipers and tumor size calculated using the equation: volume = length x width2 x 0.5. Once tumors reached 100-400 mm3 83 in size, mice were randomly allocated into one of four treatment groups each containing 8 mice. Mice were treated for 14 days as follows: group 1 received 250 jag TekdeltaFc i.p. every 3 days; group 2 received low dose cyclophosphamide in drinking water (equivalent to 30 mg/kg/day; water was changed twice weekly); group 3 received both TekdeltaFc every 3 days and low dose cyclophosphamide in drinking water; group 4 control mice received 250 jd i.p. injections of sterile PBS and untreated drinking water. Tumor growth was measured for the duration of the trial every 2-3 days. One hour prior to euthanasia, mice were injected i.p. with 150 mg/kg Hypoxyprobe-1 (Chemicon International Inc.). Mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Tumors were dissected from the surrounding tissue and cut into pieces, embedded in OCT cryomatrix and snap frozen in liquid nitrogen, fixed in 4% paraformaldehyde (USB Corporation) for 24h and paraffin- embedded, or snap frozen in liquid nitrogen and stored at -80°C for future protein and RNA isolation. Quantification of tumor hypoxic and necrotic areas Paraformaldehyde-fixed paraffin-embedded 8 |xm thick sections were de- paraffmized and sodium citrate antigen retrieval (as described in Appendix II) was performed. Following antigen retrieval, sections were washed and blocked first with Dako protein-free block (Dako) for 15 minutes, and then with 5% normal goat serum (Sigma-Aldrich) for 30 minutes. Next, sections were washed and covered with 3% hydrogen peroxide (Fisher Scientific) for 15 minutes to block endogenous peroxidase activity. Sections were washed and incubated in mouse Hypoxyprobe-1 antibody 84 (Chemicon International) (1:50) overnight at 4°C, followed by goat anti-mouse biotinylated secondary antibody (1:200) for 30 minutes. Sections were washed and treated with R.T.U Vectastain Elite ABC reagent (Vector) for 30 minutes followed by incubation with substrate reagent Diaminobenzidine (DAB) for 2 minutes. Sections were then rinsed with water, counterstained using Mayer's hematoxylin solution (diluted 1:1 with water) (Sigma-Aldrich) for 1 minute then dehydrated and mounted using Aquapolymount (Polyscience). In total, two blocks from each of twenty tumors were evaluated (five from each of the four treatment groups). Images of the tumor xenograft sections were captured in a blinded fashion using using 20X magnification objective of a Leica DMLB compound light microscope fitted with a Q imaging QICAM fastl394 digital camera using Q-Capture software. Images were merged using Adobe Photoshop 7.0 (Adobe). Depending on the size of the tumor; certain sections were subdivided into one to four fields of view. Optimas 6.0 (Optimas) software (Houston) was used to quantify areas of hypoxia and necrosis in each section which were latter expressed as percentage of hypoxia/necrosis or hypoxia and necrosis per section area. For immunofluorescent staining of the tumor xenografts, 8 (j.m thick cryosections were cut using a cryostat adjusted to -20°C. Sections were air-dried at RT, fixed in cold methanol/acetone (50:50) for 10 min at -20°C and then air-dried again. Sections were then rehydrated in PBS and blocked using 10% normal goat serum (Sigma-Aldrich) for lh at room temperature followed by mouse anti-Tie2 (1:100; BD Biosciences) for lh at room temperature and incubation with goat anti-mouse Cy3 conjugated secondary antibody for 20 minutes (1:200; Jackson ImmunoReserach). Sections were blocked using Dako protein-free block (Dako) for 15 minutes, incubated overnight at 4°C using rat anti- 85 CD31 antibody (1:50; Hycult Biotechnlogy), followed by Streptavidin conjugated to Alexa350 (1:150; Invitrogen) for 40 minutes. Hypoxyprobe antibody (Hypoxyprobe) was directly conjugated to green fluorescent Alexa488 using mouse antibody labeling kit according to manufacturer's protocol (Invitrogen; see Appendix II for details). Slides were incubated in primary antibody solution overnight at 4°C. Next day, slides were then washed briefly in water and mounted using Flourescent Mounting media (Dako). Quantification of Tie2 expression and microvessel density To evaluate Tie2 expression patterns as well as determine microvessel density, two tissue blocks from each tumor were assessed. Cryosections, 8 jim thick, were cut using a cryostat adjusted to -20°C. Once cut, sections were stored at -80°C until further use. For immunofluorescent staining, sections were air-dried at RT, fixed in cold methanol/acetone (50:50) for 10 min at -20°C and then air-dried. Then sections were rehydrated in PBS and blocked using 10% normal goat serum (Sigma-Aldrich) for lh at room temperature followed by mouse anti-Tie2 (1:100; BD Biosciences) for lh at room temperature and incubation with goat anti-mouse Cy3 conjugated secondary antibody for 20 minutes (1:200; Jackson ImmunoResearch). Sections were blocked using Dako protein-free block (Dako) for 15 minutes, incubated overnight at 4°C using rat anti-CD31 antibody (1:50; Hycult Biotechnlogy), followed by donkey anti-rat FITC secondary antibody for 30 minutes (1:100; Jackson ImmunoResearch) and 2 minutes in DAPI (4',6- diamidino-2-phenylindole) (Dako) nuclear stain. Slides were then washed briefly in water and mounted using Fluorescent Mounting media (Dako). 86 Entire sections were examined by epifluorescent microscopy using the 20X objective in a semi-blinded fashion, as previously described. Microvessel density was determined by dividing the total number of blood vessels per field of view, to obtain a value expressed as number of blood vessels per mm2. Area per field of view was calculated by capturing an image of a micrometer at a 20X-objective. Quantification of blood vessel pericyte coverage To evaluate the degree of pericyte coverage of tumor blood vessels, two blocks from each tumor were assessed. Cryosections, 8 |im thick, were fixed as previously described, then rehydrated in PBS, permeabilized using 0.05% Tween-PBS for 10 minutes and blocked using 10% normal goat serum (Sigma-Aldrich) for 30 minutes at RT. Sections were incubated in a mixture of rabbit anti-PDGFRP (1:100; Cell Signaling Technology) and biotinylated rat anti-CD31 (1:60; Hyc-ult) primary antibodies overnight at 4°C. Sections were washed in PBS and incubated in a mixture of goat anti-rabbit secondary antibody conjugated to Cy3 (1:200; Jackson ImmunoResearch) and Streptavidin conjugated to Alexa350 (1:150; Invitrogen) for 40 minutes at room temperature. Then, slides were washed in PBS and incubated in rabbit anti-desmin primary antibody conjugated to Alexa488 (1:100; Abeam, Cambridge, MA) for 30 minutes, followed by mounting in Fluorescent Mounting Media (Dako). Desmin was directly conjugated to green fluorescent Alexa488 using the rabbit antibody labeling kit according to manufacturer's protocol (Invitrogen; see Appendix II). Sections were examined using epifluorescent microscopy as previously described capturing 5 random fields. Stained blood vessels were enumerated as 87 CD3 l+/desmin+/PDGFR.p+ or CD31 +/desmin7PDGFRP" and expressed as percentage per section. Measurement of VEGF levels in tumor xenograft lysates A commercially available human VEGF ELISA kit (R&D Systems) was used to quantify VEGF levels in lysed tumor xenografts from each of the treatment groups (control, TekdeltaFc, LDM CTX, and TekdeltaFc+LDM CTX). Briefly, frozen tumor pieces (3-4 per group) were defrosted on ice and lysed using manual grinder and cell lysis buffer (Cell Signaling). Protein was collected as previously described and VEGF ELISA performed as suggested in manufacturer's instructions. Measurement of Thrombospondin-1 levels in conditioned media Malignant melanoma cell lines WM115 and WM239 were grown to 75-100% confluence and culture medium was switched to a serum-free, phenyl red-free DMEM medium (containing 1 mmol/L sodium pyruvate, and 50 jig/mL gentamicin) for 48 hours. The supernatants were then transferred into Amicon Ultra Centrifugal Filters (Millipore) and centrifuged at 4000 x g for 15 minutes at 4°C, to concentrate the conditioned media. The filters were subsequently placed on ice, and 200 JJL of the supernatant was aliquoted and stored at -80°C for western blotting analyses described previously. Statistical Analysis Calculation of preliminary summary statistics such as mean, standard deviation and standard error as well as graphing all the data was completed using Microsoft Excel (Microsoft). On all samples, Grubbs' test, also called the ESD method (extreme 88 studentized deviate), was used to determine significant outliers. Once outliers, if any, were identified and omitted from the analysis, ANOVA was perform to determine the significance within and between groups (p<0.05). Further, least significant difference (Tukey) test was used if there is significant difference between groups. Data were presented as mean, standard error and range. 89 RESULTS The effects of Tie2 inhibitor and 4HC on survival and cord formation on Matrigel of endothelial cells in vitro A significant decrease (p<0.05) was observed in the number of viable cells 24, 48 and 72h after treatment with TekdeltaFc alone and in combination with 4HC, compared to controls (vehicle only) (Figure 14). In the TekdeltaFc treatment group, viability was most significantly affected 48h after exposure (~ 45% viability). Treatment with 4HC significantly reduced endothelial cell viability at 48 and 72h (viability averaged at 52.5%), while the combination of two antiangiogenic therapies decreased the number of viable cells by 52% on average in the first 24h, and further to about 20% of control on average at 48 and 72h. TekdeltaFc interfered with the formation of tube-like structures on Matrigel in a dose dependent fashion (Figure 15) and also caused regression of already formed structures 24 and 48 hours later (Figure 16). Effects of LDM CTX and Tie2 inhibitor in xenograft model Metronomically administered low dose CTX alone and in combination with TekdeltaFc had no significant effect on tumor growth in human melanoma xenografted to immune deficient mice after two weeks of treatment (Figure 17). However, both therapies (TekdeltaFc and LDM CTX) alone showed significant differences from control (p<0.05) in growth at earlier time points (day 3, 7 and 10). Alone, TekdeltaFc caused the greatest decrease in tumor volume, followed by LDM CTX. Surprisingly, combined therapy had no effect on tumor growth compared to control. 90 There were no significant differences in amount of hypoxic or necrotic areas, individually or combined, between different treatment groups (Figure 18A and 18B). However, significantly lower amounts of hypoxia and necrosis compared to viable tissue were observed in TekdeltaFc and 4HC treatment groups than in control and combined group. We quantified blood vessel density (BVD) as well as the proportion of Tie2 negative vessels in tumor xenograft sections stained for CD31 and Tie2 (Figure 19). Statistical analysis showed no difference in the proportion of Tie2 negative blood vessels between treatment groups (Figure 20A). The average number of Tie2 negative blood vessels was about 12.5 %. Although decrease in BVD was observed in LDM CTX treated tumors compared to TekdeltaFc treatment group, this was not statistically significant (p>0.05) (Figure 20B). Tie2 inhibitor significantly increased pericyte coverage of tumor blood vessels compared to low dose CTX treatment Triple immunofluoresence was utilized on frozen xenografted sections using desmin and PDGFRP to detect vascular mural cells (Guillemin and Brew, 2004). Vessels were categorized as desmin or PDGFRP positive or negative (Figure 21). We observed a statistically significant difference (p<0.05) between treatments groups, with the most pericyte coverage in vessels of the TekdeltaFc treatment group, and the least pericyte coverage in vessels of the LDM CTX group. In fact, TekdeltaFc tumors had statistically significant (p<0.05) increased pericyte coverage compared to all other groups (Figure 22). 91 TekdeltaFc and LDM CTX increase VEGF expression Tumor pieces from each treatment group were lysed and VEGF expression analyzed, showing significant increase (p<0.05) of VEGF concentration in TekdeltaFc and LDM CTX combined treated tumors compared to all of the other treatment groups. In addition, VEGF concentration was significantly higher (p<0.05) in LDM CTX compared to control group (Figure 23). The effects of Tie2 inhibitor and 4HC on Tie2 signaling transduction in vitro Lower level of Tie2 phosphorylation at Tyr992 was observed in cultured BAECs at 24 hours after both TekdeltaFc and 4HC treatments (Figure 24B), but not at earlier time points (Figure 24A). Akt phosphorylated at Ser473 did not change at any time point. Interestingly, when cells were treated with both TekdeltaFc and 4HC combined, Tie2 phosphorylation increased after 24 hours of treatment compared to TekdeltaFc and 4HC treatment groups (Figure 24B). Hypoxia induces downregulation of Tie2 expression in vitro In order to get insight into the effect of tumor microenvironment conditions, such as hypoxia, hypoglycemia, and cancer cell secreted molecules, on Tie2 expression, we exposed BAECs to severe hypoxic (<0.1% O2), hypoglycemic (glucose free media) and cancer cell conditioned media in vitro (Figure 25). We observed a significant (-40 %) decrease (p<0.05) in Tie2 protein expression 12 and 24 hours after treatment with hypoxia. Hypoglycemia caused transient downregulation of Tie2 observed only 12 hours after treatment. There was no significant change in the expression of Tie2 receptor 6h post either hypoxic or hypoglycemic treatment, and combination of both conditions 92 showed decreased Tie2 expression by about 60% at both 12 and 24h (Figure 25A and 25B). When compared to control, BAECs cells showed downregulation of Tie2 expression when treated with serum free conditioned medium from either human melanoma (WM239) or colorectal cancer (HCT116) cells for 24h, suggesting that tumor cell secreted factors may play a role in downregulation of Tie2 receptor in this model (Figure 25C) but that these effects are not tumor type specific. There was no evidence of a dose response when administering varying amounts of conditioned media. When investigated in vivo, Tie2 expression patterns in xenografted tumors turned out to be complex - both Tie2 positive and Tie2 negative blood vessels were observed in hypoxic areas of human melanoma mouse xenografts, with no apparent pattern linking ischemia and expression levels (Figure 26). TSP-1 levels are modulated by LDM CTX and hypoxia Basal levels of expression of TSP-1 in both primary and metastatic melanoma cell lines were investigated. There was a significant decrease (p<0.05) of about 97% in TSP-1 protein between cells isolated from primary and secondary melanoma lesions (Figure 27A). In addition, when both primary (WM115) and metastatic (WM239) derived melanoma cells were treated with 4-HC in normoxic and hypoxic conditions, we observed an increase in the expression of TSP-1 (Figure 27B). Furthermore, hypoxia downregulated levels of TSP-1 in the primary melanoma cell line WM115. Metastatic cell line WM239 expressed such low levels of TSP-1 that it was virtually undetectable by western blot in both normoxic and hypoxic protein collections (results not shown). 93 DISCUSSION In vitro, TekdeltaFc and 4-HC, alone and combined caused significant decrease in survival of normal endothelial cells. The mechanisms of action of TekdeltaFc in our model likely involved the inhibition of endothelial survival pathways through binding to both angiopoietin 1 and 2, as well as Tie2, which therefore prevented downstream signaling. The metabolically active form of cyclophosphamide, 4-HC, was cytotoxic to endothelial cells, as reported in previous studies (Moore, 1991). TekdeltaFc also caused regression of already formed endothelial, cord-like structures on Matrigel and prevented new cord formation, consistent with its mechanism of inhibition of Tie2 survival pathways (Jones and Dumont, 2000; Lin et al, 1997). Although we did not look at this in our studies, the mechanism of action of TekdeltaFc (the extracellular domain of murine Tie2/Tek receptor fused to the Fc portion of murine IgG) has been previously suggested by other researchers. When used by Das et al, TekdeltaFc inhibited retinal neovascularization by 87%, by binding, with high avidity, to both Angl and Ang2 (Das et al., 2003). In fact, many researchers used Tie2 extracellular domain to design inhibitors that will bind to it and to angiopoietin ligands, in order to interfere with signaling of this pathway and its inhibition. As such, some studies used injections of adenovirus expressing the extracellular domain of Tie2 (Hangai et al., 2001) or injections of soluble Tie2 receptor Fc fusion protein (Lin et al., 1997; Takagi et al., 2003), resulting in various amounts of inhibition of retinal and tumor revascularization, depending on the model used. These differences may be due to the dosage, the protein concentration, binding activity or stability of different proteins. 94 Another similar inhibitory molecule, ExTek, was shown to function as a very potent inhibitor of Tie2 through two mechanisms of action - by sequestering available angiopoietin, and by binding to Tie2 receptor, inhibiting phosphorylation and downstream signaling molecules related to cell survival, similar to TekdeltaFc (Hangai et al., 2001; Lin et al., 1997; Peters et al., 2004). Interestingly, ExTek decreased the number of lung metastasis in a murine melanoma model (Lin et al., 1998a). Other studies employing different versions of the Tie2 extracellular domain, used as an inhibitor, achieved similar effects in different tumor models (Siemeister et al., 1999; Stratmann et al., 2001; Tanaka et al., 2002). In vivo, both TekdeltaFc and LDM CTX alone caused significant decreases in tumor volume of xenografts at earlier time points, but they failed to do so after two weeks of treatment. This could be due to the fact that most advanced malignant tumors produce multiple growth factors, and so targeting only angiopoietins by using TekdeltaFc, may not be adequate for complete tumor control (Carmeliet and Jain, 2000; de Jong et al., 1998; Kerbel et al., 2001; Yancopoulos et al., 2000). In addition, in our model, tumors may have become resistant to both TekdeltaFc and LDM CTX, allowing them to re-grow after initial inhibition. Moreover, the presence of Tie2 negative blood vessels in these tumors did not significantly impact the effectiveness of this antiangiogenic trial suggesting that presence of these vessels had no obvious effect on trial outcome. TSP-1 can directly affect endothelial cell migration and apoptosis as well as influence VEGF expression (Greenaway et al., 2007; Jimenez et al., 2000; Lawler, 2002; Nor et al., 2000; Straume and Akslen, 2001). When we tested the metastatic-derived melanoma cells WM239 in vitro, we observed drastic reduction of basal expression of TSP-1 95 compared to the primary lesion derived WM115 cells, which increased when these cells were treated with 4-HC. This was not surprising since human melanoma cell have been shown to synthesize and secrete TSP-1 (Apelgren and Bumol, 1989; Trotter et al., 2003; Varani et al., 1989). Decreased TSP-1 expression has been associated with malignant progression (Zabrenetzky et al., 1994). Decrease in TSP-1 observed with malignant progression, as seen in our model, may be due to mutations of p53 which positively regulates TSP-1 gene promoter sequences in wild-type p53 (Grant et al, 1998). In addition, independent of p53 status, metastatic melanoma lesions usually contain areas of hypoxia (Lartigau et al., 1997) characterized by high levels of VEGF and low levels of TSP-1 (Laderoute et al., 2000). 4-HC did cause an increase in the production of TSP-1 in WM115 melanoma cells grown in normoxia and hypoxia. Therefore, in agreement with previous reports (Bocci et al., 2003; Hamano et al., 2004) our data suggests that LDM CTX in our mouse model had an ability to induce TSP-1 expression but this did not translate into VEGF decrease - in fact levels of VEGF increased in LDM CTX group compared to control group. Decrease in VEGF may have occurred at earlier time points but may have eventually been overridden by anti-tumor resistance to LDM CTX. Angiopoietin-2, commonly overproduced by many cancers including melanomas, destabilizes blood vessels by causing the detachment of pericytes (Yancopoulos et al., 2000). It has pro-angiogenic properties in the presence of angiogenic factors such as VEGF (Holash et al., 1999; Peters et al., 2004; Yancopoulos et al., 2000). As seen in our study, tumors in the LDM CTX group had significantly lower amount of pericyte covered blood vessels, consistent with them going through the process of angiogenesis or vessel 96 regression. Since VEGF levels are increased in these treated tumors, such pericyte free vessels were likely in the process of regression initially, but according to the VEGF levels observed in these treated tumors it is more likely these are in the process of angiogenesis at the time of tumor excision. We also observed that TekdeltaFc alone caused significant increases in pericyte coverage of blood vessels compared to control, LDM CTX or combination therapy. This is not surprising since antiangiogenic therapies have been previously shown to improve chemotherapy by inducing maturation of the blood vessels via increasing pericyte coverage (Lin and Sessa, 2004). In abnormal tumor vasculature, VEGF (which is abundantly produced by WM239 melanoma cells, reported in Chapter 2) induces expression of Ang-2 from endothelial cells in the microvasculature (Harfouche and Hussain, 2006; Lobov et al., 2002). By binding to Tie2, Ang-2 becomes an autocrine regulator of endothelial cell function and its action as either agonist (Harfouche and Hussain, 2006; Teichert-Kuliszewska et al., 2001) or antagonist (Maisonpierre et al., 1997) is context dependent (Eklund and Olsen, 2006). It is now known that, in culture, Ang-2 inhibits the stabilizing effects of Angl but weakly activates Tie2 if Angl is absent (Yuan et al., 2009). In our tumor model, TekdeltaFc likely caused sequestering of abundantly expressed Ang-2, thus allowing Ang-1 to bind and phosphorylate Tie2, hence the observed increase in pericyte coverage. Interestingly, tumor xenografts treated with TekdeltaFc and LDM CTX combination therapy grew at the same rate as controls. Seemingly these two therapies "cancel" each other. This is possibly due to the non-sufficient production of Angiopoietin-1 pointing to the possibility that TekdeltaFc in this trial group may have bound to Tie2 receptor, 97 prevented Tie2 phosporylation and hence destabilized the blood vessels. It remains to be seen whether TSP-1 plays a role in the inhibition of Ang/Tie2 signaling, and hence blood vessel stability. Although TekdeltaFc caused decreased phosphorylation of endothelial Tie2 receptor in vitro, as expected, so did 4-HC. This suggests that these compounds can have a direct or indirect effect on Tie2 receptor activation. When these therapies are combined in vitro, the phosphorylation of Tie2 receptor was enhanced compared to that of control endothelial cells, which agrees with the tumor growth effects we observed in vivo. This data supports the idea that these two different antiangiogenic therapies have opposing effects. Future studies are aimed at elucidating the mechanism of this action. Although previous studies have observed an upregulation of Tie2 gene expression and protein levels in endothelial cells under mild hypoxia (-2% C^XNilsson et al., 2004; Oh et al., 1999), there have been no reports on the effect of severe hypoxia, which is most commonly observed in solid tumors. Therefore, the culture conditions used in these experiments may be a better representation of the in vivo conditions encountered in human tumors. In addition, increase in levels of hypoxia may be one of the reasons why TekdeltaFc initially caused significant anti-tumor effects that were not sustained. Our studies demonstrate that hypoxia decreases Tie2 expression by endothelial cells in vitro, which coincides with our in vivo studies where the amount of hypoxia matched the amount of Tie2 negative blood vessels observed in our tumors. In spite of this observation, there was a complex relationship between hypoxic regions and Tie2 expression by tumor vasculature. Receptor down-regulation is considered to be a primary mechanism for the attenuation of downstream signal transduction, resulting in an overall 98 loss of receptors from the cell surface, thereby diminishing the ability of a cell to respond to further stimulation. This phenomenon has been readily observed for numerous tyrosine kinase receptors such as platelet-derived growth factor receptor (Keating and Williams, 1987), insulin receptor (Kasuga et al., 1981) as well as Tie2 receptor (Bogdanovic et al., 2009; Bogdanovic et al, 2006). It has also been suggested that internalization of activated receptors may be necessary to facilitate downstream signaling and that a proper signaling response may require receptor endocytosis (Sorkin and Waters, 1993; Wiley and Burke, 2001). These studies were designed to provide insight into how the combination of two antiangiogenic therapies, in this case targeted and non-targeted, affects the tumor growth response in malignant melanoma. Surprisingly, we found that while LDM CTX or TekdeltaFc alone induced significant suppression in tumor growth in early phases of the trial, the same agents in combination did not. This was despite the fact that such combined therapy exhibited synergistic effects on endothelial cells in vitro. Although combination of antiangiogenic therapies is becoming a common practice (Colleoni et al, 2006; Dellapasqua et al., 2008; Garcia-Saenz et al., 2008; Jurado et al., 2008), our studies provide evidence that doing such may not always result in synergistic or additive outcomes and that caution should be taken in designing such combinations. 99 1.20 •24 •48D72h 1.00 0.00 control TekdeltaFc 4HC TekdeltaFc+4HC Treatments Figure 14. Endothelial cell survival after treatment with TekdeltaFc and 4-hydroxycyclophosphamide (4-HC) in vitro. Enumeration of viable cells 24, 48 and 72 hours post treatment with TekdeltaFc alone and in combination with 4-HC reveals significant (*p < 0.05) decrease in survival of BAECs. 100 Figure 15. Effects of TekdeltaFc on formed endothelial cell cords on Matrigel. Images on the left show BAECs plated on Matrigel before treatment with TekdeltaFc and the images on the right show the same field 24h after TekdeltaFctreatment. Arrows indicate the area of interest, depicting tube like structures formation or regression. Scale bar is 100 jum. 101 Treatment for 24h Treatment for 48h Figure 16. Endothelial cell cord formation on Matrigel in the presence of TekdeltaFc in vitro. TekdeltaFc treatment was added concurrent with seeding of the cells on Matrigel. Images on the left are of representative fields taken 24h after seeding and treatment initiation. Images on the right are of the same fields 48h later. Arrows represent area of interests depicting a particular tube-like structure formation or regression. Scale bar is 100 pun. 102 V lOOOi 3E 900" 800" control 9 700" TekdeltaFc 3S 600" LDM CTX S 500" 01 400" TekdeltaFc+ s01 ) 300" LDM CTX £u OJ 200" > eg 1UU o f OS 0 + -1 1 I— -i— -1 6 8 10 12 14 16 Day of treatment day 3 day 7 day 10 + + Control 167.1 ± 25.7 309.8 ± 37.8 * 474.2 ± 44.7 * TekdeltaFc 109.3 ± 23.5* 180.7 ± 35.4* 276.6 ± 72.7* LDM CTX 137.5 ± 26.1 219.7 ± 17.1 + 334.6 ± 23.2+ TekdeltaFc+ LDM CTX 179.3 ± 19.3* 277.9 ± 59.7 419.5 ± 65.2 Figure 17. In vivo xenografts of human melanoma cancer treated with TekdeltaFc and low dose metronomic cyclophosphamide. Relative tumor growth for each treatment group - control, TekdeltaFc, low dose metronomic cyclophosphamide and combination for two weeks. Table shows statistical significance between treatment groups - significantly different groups are marked by either asterisk (*) or by plus (+). 103 B 90.00n D viable tissue "hypoxic tissue 80.00 n CS necrotic tissue 2 70.00 D hypoxic + C necrotic tissue .2 60.00 o C/4)5 50.00 Figure 18. Quantification of hypoxic and necrotic areas in tumor xenografts treated with TekdeltaFc and LDM CTX. (A) Representative low (5X) and high (20X) magnification images depicting hypoxia immunostaining of hypoxyprobe adducts and necrosis (degenerated tissue) in tumor sections. Dark brown areas are hypoxic (arrow), light brown areas are viable tumor (arrowhead), and areas lacking color are necrotic (asterisk). (B) Quantification of the average percentage of hypoxia, necrosis or both in each treatment group, shows significant differences in ratios of viable tumor areas to necrotic and hypoxic areas in TekdeltaFc and LDM CTX treatment groups (*p < 0.05). Scale bar is 100 jum. 104 / * / / * / / - / Figure 19. Dual immunofluorescent staining of melanoma xenografts for CD31 and Tie2. Tie2 is fluorescently labeled red using Cy3 flourochrome while CD31 is fluorescently labeled green using FITC conjugate. The last image is an overlay of red and green channels. Representative Tie2 positive blood vessels are marked with an asterisk while representative Tie2 negative blood vessels are labeled with arrows. Scale bar is 100 pim. 105 £ 18.00' 116.00' 114.00 J12.C S 10.001 ® 8.00" m 6.00' = 400 1 P 2.00 J 0.00 Control TekdeltaFc LDM CTX TekdeltaFc +LDM CTX Treatment Groups 801 N E 70 E t- nOl. 60 >> 50 e • Figure 20. Quantifying % Tie2 negative blood vessels and blood vessel density in tumor xenografts treated with TekdeltaFc and LDM CTX. (A) Graph depicting the average % of Tie2 negative blood vessels per treatment group, where the difference between groups is not statistically significant (p > 0.05). (B) Graph showing average blood vessel densities per mm2 between treatment groups, also with no statistical significance in blood vessel density between different treatment groups tumors (p> 0.05). 106 Figure 21. Immunostaining of blood vessels and pericytes in treated tumors. (A) Images on the left show blood vessels that were immunofluorescently stained using Desmin directly labeled using Alexa 488 (green), PDGFRP tagged to Cy3 (red) and CD31 tagged to Streptavidin 350 (blue). Images on the right are overlays of desmin/CD31, PDGFR(3/cd31 and desmin/PDGFRp and CD31. Asterisk marks blood vessel negative for desmin/PDGFRp while arrow indicates blood vessel with positive desmin/PDGFRP staining. Scale bar is 100 jum. 107 Control TekdeltaFc LDM CTX TekdeltaFc Treatment + LDM CTX Figure 22. Quantifying the amount of blood vessel pericyte coverage in TekdeltaFc and LDM CTX treated tumors. The percentage of stable' blood vessels with pericyte coverage was significantly different between TekdeltaFc and all other treatment groups (*p<0.05). 108 a,b,c a,c —f- a -f" Control TekdeltaFc LDM CTX TekdeltaFc+ LDM CTX Figure 23. Expression of VEGF (pg/ml/jig) in tumor xenograft lysates. Concentration of VEGF measured by ELISA shows significant differences in VEGF expression between different treatment groups. Groups with the same letters are significantly different from each other. TekdeltaFc+ LDM CTX has significantly higher expression (p<0.05) of VEGF from all other treatment groups. The lowest VEGF concentration was detected in control while the highest VEGF concentration was detected in combined group, showing that both TekdeltaFc and LDM CTX upregulate VEGF expression, both individually and combined. 109 TekdeltaFc + - + - + - + + - + - + + - + + 4-HC - - + + 160 kDa pTie2 (Tyr992) 160 kDa Tie2 pAkt (Ser473) pan Akt B TekdeltaFc - + - + 4-HC - - + + 160 kDa phosphoTie2 (Tyr992) 160 kDa •*»«••«•»«» Tie2 60kDa -mmmmmm-mm phosphoAkt(Ser473) 60 kDa Pa» Akt Figure 24. Change in native and phosphorylated Tie2 in BAEC treated with TekdeltaFc and 4-HC in vitro. (A) Western blot demonstrating no change in native Tie2, phosphorylated Tie2 at Tyrosine992, native and phosphorylated Akt at Ser473 15, 30 and 60 minutes after treatment with TekdeltaFc, 4HC and combination. (B) At 24 hours, there is decrease in phosphorylated Tie2 but not in phosphorylated Akt in Tekdelta and 4HC treatment groups. Phosphorylation of Tie2 in combined treatment group was higher compared to controls. 110 6 hours 12 hours 24 hours C LO LG LOLG LO LG LOLG LO LG LOLG Tie2 (160kDa)' a-tubulin (55kDa) 1.2 D6h B12h n24h B c e a x V 0.8 '3 * * uo Q. 0.6 > 0.4 A £ 0.2 Control Low Oxygen Low Glucose Low Oxygen Low Glucose Colorectal Control Melanoma CM Cancer CM 12 34512 3 45 12 Tie2 (160kDa) a-tubulin (55kDa) Figure 25. Expression of Tie2 receptor in hypoxia and hypoglycemia in vitro (A) Representative western blots of Tie2 protein expression in BAECs exposed to control (C), low oxygen (LO), low glucose (LG) and combined (LOLG) treatment for 6, 12 and 24h (B) Corresponding graph showing significant decrease in Tie2 protein levels (normalized to a-tubulin) after 12 and 24h of exposure to LO or combination of LOLG (p < 0.05) (C) Exposure of BAECs to cancer cell conditioned media collected from malignant melanoma cell line (WM239) and colorectal cancer cell line (HCT116) caused a decrease in Tie2 expression compared to controls (normalized to a-tubulin). Ill Figure 26. Expression of Tie2 receptor by vessels in hypoxic and normoxic areas of tumor xenografts. Panels display hypoxyprobe (green), CD31 (blue), Tie2 (red) and hypoxyprobe/CD31/Tie2 overlaid staining. Images show vessels within hypoxic regions (green) that are positive for Tie2 expression (arrows) and that are negative for Tie2 expression (arrowheads). Images also show Tie2 positive blood vessels in normoxic areas (asterisks). Scale bar is 100 jum. 112 170-180' TSP-1 kDa Amido Black Protein Staining B WM115 C C+T H H+T TSP-1 170-180- kDa Amido Black Protein Staining Y Amido Black Protein Staining Figure 27. Expression of TSP-1 in conditioned medium from human primary and metastatic melanoma cell lines (A) Western blot of basal TSP-1 protein production, detected in concentrated conditioned media of WM115 and WM239. Also depicted is respective Amido Black-stained PVDF membrane for loading normalization purposes. (B) A Western blot of TSP-1 expression by WM115 and WM239 cells, after exposure to normoxia (C), normoxia + 4-HC treatment (C+T), hypoxia (H), and hypoxia + 4-HC treatment (H+T) for 48 hours. Note the increase in TSP-1 expression after 4-HC treatment and decrease in expression when cells are placed in hypoxic environment. Respective Amido black stained PVDF membrane was also included for loading normalization purposes. 113 CHAPTER 4 - Differential expression of Tie2 and VEGFR2 receptors in clones derived from isolated bovine endothelial progenitor cells 114 INTRODUCTION A process of angiogenesis has long been thought to represent the principle paradigm for neovascularization (Hanahan and Folkman, 1996), but in the last decade, focus has shifted towards investigation of the role of bone marrow derived cells in adult neovascularization - in particular of a subset of these cells called endothelial progenitor cells (EPCs). These progenitors have the ability to mobilize from the bone-marrow into peripheral circulation, home to sites of angiogenesis, differentiate into mature endothelial cells and incorporate into the vasculature at the sites of ischemia, tumor formation and myocardial infarction (Nolan et al., 2007; Orlic et al., 2001; Shintani et al., 2001; Takahashi et al., 1999; Weber et al., 2004). However, the research findings on this subject are conflicting - possibly due to the highly variable approaches undertaken in different research venues with regard to cell source, cell purification, the animal model or assay used, method of detection and analysis, and data interpretation (Timmermans et al., 2009). Lack of a unique EPC marker makes universal identification of these cells a challenge due to important phenotypical and functional overlap between EPCs, haematopoietic cells and mature endothelial cells (Ingram et al., 2005; Ingram et al., 2004; Yoder, 2009; Yoder et al., 2007). For instance, haematopoietic-derived cells such as monocytes, granulocytes and even haematopoetic stem/progenitor cells co-express a host of similar surface markers along with endothelial cells (Yoder et al., 2007). Such bone marrow derived cells are also involved in vascular repair, making it difficult to discriminate them from each other. Therefore, the diverse cell types now known to be recruited to populate sites of 115 neovascularization may have previously been lumped into the single term 'EPC' in many early studies of postnatal vasculogenesis, explaining some of the apparent controversy in the field (Yoder, 2009). In addition, variability in identification and characterization of EPCs by different research groups may in part be due to the differences in methodology used to isolate these cells (Yoder, 2009). For instance, plating of peripheral blood mononuclear cells onto fibronectin (Asahara et al., 1997; Yoder et al., 2007) in culture medium enriched with endothelial specific growth factors gives rise to EPCs, characterized by expression of mature endothelial markers. However, others have shown that similarly isolated and cultured adherent cells expressing the above antigens may also co-express macrophage markers (De Palma and Naldini, 2006; Schmeisser et al., 2001; Timmermans et al., 2007). In contrast, plating of peripheral blood mononuclear cells on type 1 rat tail collagen yields colonies appearing within a week, that display limited proliferative potential, as well as colonies appearing 14-21 days post-plating, with high proliferative potential (Ingram et al., 2004). Researchers have also collected non-adherent cells and re- plated them on fibronectin, producing two types of colonies- endothelial cell colony forming units (EC-CFUs) and blood outgrowth endothelial cells (BOECs) (Ingram et al., 2004). Another isolation method employed involves the usage of monoclonal antibodies and fluorescence activated cell sorting to isolate and enumerate cells that express particular markers of interest (Asahara et al., 1997). Defining the precise role and contribution to tumor vasculature of these EPCs has been an intense area of research primarily due to important implications for novel anticancer therapy as well as their applications for other diseases (Hristov and Weber, 116 2004) such as myocardial ischemia. In fact, transplantation of isolated and ex vivo expanded human EPCs improved blood flow recovery and capillary density in several animal hind-limb ischemia models (Kalka et al., 2000a) and improved ischemic heart conditions (Kawamoto et al., 2001). Initial clinical trials using autologous progenitor cell transplantation are underway, showing great promise for patients with ischemic limbs as a consequence of peripheral arterial disease (Mund et al., 2009; Smadja et al., 2007; Takahashi et al., 2009; Tateishi-Yuyama et al., 2002). Here, we investigated if progenitor cells isolated from circulating peripheral blood mononuclear cells can give rise to endothelial linage, and if their differentiated phenotype can be influenced by maturation on different extracellular matrices. In addition, we investigated the expression profiles of their endothelial markers that were differentially expressed, and used the Matrigel plug model to determine if differential expression of some endothelial receptor tyrosine kinases by these cells influenced homing to sites of neovascularization in vivo. These studies have important implications in our understanding of EPCs, and potentially impact cancer therapeutics designed to target specific endothelial markers. 117 MATERIAL AND METHODS A list of all chemicals and suppliers as well as the description of materials and solutions prepared for the following experiments are detailed in Appendices I and II, respectively. Buffy-Coat Preparation All procedures described below were done according to the guidelines and recommendations of the Canadian Council of Animal Care and approved by the University of Guelph Local Animal Care Committee. Peripheral blood samples (approximately 100 ml per isolation) were collected from healthy cows (Veterinary Teaching Hospital, University of Guelph) using vacutainers (BD Biosciences, Mississauga, ON, Canada) containing acid citrate dextrose (ACD) solution. Samples were centrifuged at 1200 x g for 20 minutes to separate blood. Buffy-coat mononuclear cells (MNCs) were collected along with some red blood cells that were lysed for 4 minutes with 4M Ammonium Chloride Solution (detailed preparation protocol can be found in Appendix II). Cells were spun at 350 x g for 4 minutes to obtain a pellet and washed twice with HBSS (Invitrogen). Finally, the pellet was resuspended in complete EGM-2MV (components listed in Appendix II). Mononuclear cells were counted using a haemocytometer. Four percent Trypan Blue (Sigma) was also used to identify dead cells and debris. 118 Culture of MNCs to obtain EPC derived colonies To investigate whether plating of isolated blood MNCs cells on different extracellular matrices had an effect on the number of CEP derived colony "islands" that formed, fresh isolated bovine MNCs were seeded at a density of 5000 cells/cm2 into separate plates pre-coated using 10 (ig/ml of fibronectin (Sigma) or 5 |ig/cm2 type IV rat tail collagen (BD Biosciences), or left uncoated to serve as a control. Collagen was prepared by adding 4.2 mL of type IV rat tail collagen to 1.2 mL of 7.5% Sodium Bicarbonate Solution (Invitrogen) and 0.6 mL of 10 X Minimal Essential Medium (Invitrogen). 1 mL of the solution was added to each well of a 6-well plate. The plate was incubated for 20 minutes at room temperature to allow the collagen to solidify into a gelatinous state. Similarly, plates were pre-coated with fibronectin (Sigma) that was diluted using sterile PBS solution (Sigma) to 10 (ig/ml. Thirty minutes after the solution was added, PBS residue was removed, and plates were washed twice with fresh sterile PBS and used. All plates were maintained at 37°C, 5% CO2, in a humidified incubator. Media was changed every 3-4 days and non-adherent cells were discarded. Mononuclear colony "islands" were enumerated under 10X magnification with inverted phase contrast microscope. In a similar experiment, we investigated whether the removal of non- adherent cells and the media 3, 5 or 7 days after incubation of MNCs in normoxia, or 7 days after incubation of MNCs in hypoxia (<0.01% O2) , affected the final number of CEP colony "islands" obtained. For further experiments, seeding of 40 x 106 MNCs /100 mm tissue culture dish was used. Mononuclear cells were seeded onto either collagen, fibronectin or non-coated 119 plates. Once media was replaced 3 days after the initial plating, colonies appeared anywhere from 4-21 days later. While the colonies were still small enough to remain physically distinct, they were released from the original plate using cloning disks (Sigma) dipped in trypsin, and seeded into separate wells of non-coated 48-well tissue culture plates and grown in complete EGM-2 MV until confluence was reached. At this point, clones were expanded for further characterization and studies. Western Blot Analysis In order to characterize the clones obtained, cells were grown to confluence and lysed using cell lysis buffer (Cell Signaling) (detailed list of components listed in Appendix II). Proteins were separated using SDS-PAGE on 7.5% polyacrylamide gel, transferred to a PVDF membrane (Roche) using wet transfer, blocked using 5% milk for 1 hour and probed overnight using either goat anti-CD31 (1:1000, Santa Cruz), rabbit anti-VEGFR2 (1:1000, Cell Signaling), mouse anti-N-cadherin (1:2500, BD Pharmingen), mouse anti-a-smooth muscle actin (1:1000, Sigma), rabbit anti-PDGFR|3 (1:1000, Cell Signaling), mouse anti-Tie2 (1:1000, Cell Signaling) or mouse anti- VEGFR1 (1:1000, AbCam). Mouse anti-a-tubulin (1:400,000, Sigma) was used for normalization purposes. Appropriate anti-mouse (1:40,000, Sigma), anti-rabbit (1:10,000, Sigma) or anti-goat (1:40,000, Sigma) secondary POD antibodies were used. Bands were visualized using Chemilluminescence detection kit (Roche), exposed to X-ray film and quantified by densitometry. 120 Fluorescent Labeling Using Cell Tracker Probes To be able to visualize injected cells, clones of EPC derived endothelial cells chosen based on variable VEGFR2 and Tie2 expression were labeled in vitro using CellTracker Red CMPTX (577/602 rail) Kit (Invitrogen) to produce red fluorescent cells. Immediately prior to labeling, CellTracker Red CMPTX fluorescent probe was diluted in PBS to a previously optimized working concentration of 5 jiM. Confluent cells were washed in PBS and exposed for 15 minutes to pre-warmed diluted fluorescent probe at 37°C. Solution was then removed and replaced with DMEM (Sigma Aldrich) supplemented with 10% fetal bovine serum, 50 ng/ml gentamicin and Immol/L sodium pyruvate for at least 30 minutes. Cells were trypsinized, counted and resuspended to a concentration of 2 x 106 cells/50 [i\ of sterile 0.1% BSA/PBS solution. In vivo Matrigel Plug Assay Twenty five female athymic nude mice (Charles River Canada) were injected subcutaneously with 500 jj.1 of ice-cold Matrigel (BD Biosciences) premixed with 5 jig/ml of mouse VEGF (Roche) to initiate a strong angiogenic response. After 21 days, each mouse was injected into the tail veil with 50 pi of cell suspension containing 2 x 106 cells. Each fluorescently labeled endothelial clone was injected into 5 mice, with a total of 5 different clones injected. After 3 days, mice were euthanized by CO2 chamber asphyxia and cervical dislocation. Matrigel plugs were removed as well as pieces of lung, liver and kidney, placed in OCT cryomatrix and snap frozen in liquid nitrogen for further analysis. 121 Visualization of injected cells Ten micron thick cryosections were obtained from Matrigel plugs, lung, liver and kidney. Sections were briefly air dried and fixed in 4% paraformaldehyde for 15 minutes at room temperature, followed by washing in PBS. To visualize the cellular nuclei, sections were stained with DAPI for 2 minutes and mounted using Aquapolymount (Polyscience) and visualized using an epifluorescent microscope (Olympus) as described previously. Imaged fields were chosen randomly. For CD31 staining, fixed cryosections were blocked for 30 minutes using Dako protein block (Dako) and incubated for lh with rat CD31 antibody (dilution 1:100) (Hyclone). After washing away the primary antibody with PBS (3 times 5 minutes) sections were incubated in goat anti-rat FITC secondary antibody (dilution 1:200) (Jackson Laboratories) for 30 minutes. Statistical analysis Calculation of preliminary summary statistics such as mean, standard deviation and standard error as well as graphing all the data was completed using Microsoft Excel (Microsoft). On all samples, Grubbs' test, also called the ESD method (extreme studentized deviate), was used to determine significant outliers. Once outliers, if any, were identified and omitted from the analysis, ANOVA was perform to determine the significance within and between groups (p<0.05). Further, least significant difference (Tukey) test was used if there was significant difference between groups. Data were presented as mean, standard error and range. 122 RESULTS Phenotypic differences in cells arising from bovine peripheral blood MNCs Adherent cells yielded colonies of two distinct phenotypes that appeared between days 4-21: mononuclear cell "islands" resembling CFU-EC colonies and characteristic cobblestone endothelial colonies (Figure 28A). The phenotype of these colonies did not appear to depend on either the time to outgrowth (4-21 days) or the matrices (collagen, fibronectin or none) on which they were grown. Although colonies readily grew on all plates - with no coating, with fibronectin or collagen - the number of mononuclear cell "islands" obtained from fibronectin and collagen coated plates was significantly higher than in plates that were not coated with any extracellular matrix (Figure 28B). CFU- ECs-like cell islands failed to form secondary colonies when transferred to a 48 well plate using cloning disks and were therefore unable to be studied further. Cells of endothelial phenotype had an ability to attach to the plates, proliferate and be further passaged. We found that removal of non-adherent cells 3, 5 and 7 days after initial seeding of MNCs did not significantly increase the number of mononuclear cell "islands" and endothelial colonies observed (Figure 28C). Some studies suggested that increased number of EPC colonies arise in cultures that are hypoxic pre-conditioned (Akita et al, 2003). Maintenance of plates for 7 days in hypoxic conditions after plating did not produce any significant difference in the number of attached cellular "islands" (Figure 28C). 123 Characterization of expanded colonies reveals differential expression of receptor tyrosine kinases Protein lysates were used to characterize the MNC derived clones using the endothelial markers Tie2, N-cadherin, VEGFR2, VEGFR1 and CD31, as well as a- smooth muscle actin and PDGFRP to rule out presence of fibroblasts or other stromal cells (Figure 29). Western blotting revealed that EPC derived endothelial clones were strictly expressing only endothelial markers. Only some endothelial clones expressed N- cadherin but the significance of this has yet to be determined. Plating of MNC on fibronectin, collagen or uncoated dishes did not seem to impact receptor tyrosine kinase expression patterns of resultant endothelial clones (Figure 29). Importantly, many clones with typical endothelial morphology had markedly different expression of endothelial receptor tyrosine kinases (RTKs) such as Tie2 and VEGFR2 (Figure 30A). Therefore, clones were identified that had strong expression of both Tie2 and VEGFR2, weak Tie2 and VEGFR2 expression, weak Tie2 and strong VEGFR2 expression, and strong Tie2 and weak VEGFR2 expression. Although their expression profiles of Tie2/VEGFR2 were different, morphologically these cells did not appear any different from each other (Figure 30B). When subsequently tested, randomly chosen clones showed no expression of CD45, a pan haematopoietic marker commonly expressed on lymphocytes, mature myeloid cells and haematopoietic progenitor cells (Figure 31). All cells expressed CD31 in various amounts, showing that they are of endothelial origin. Clones with differential RTK expression were also plated on Matrigel to assess their ability to form tube-like structures. Twenty four hours post plating, clones 124 expressing high levels of Tie2 and low levels of VEGFR2 and vice versa formed cord- like structures in Matrigel, while a clone with low Tie2 and VEGFR2 expression did not (Figure 32). Clones with differentia] expression of Tie2/VEGFR2 home to sites of angiogenesis in Matrigel plug assay in vivo We investigated if different MNC derived endothelial clones are able to home to sites of active angiogenesis and integrate into newly formed blood vessels in Matrigel plugs subcutaneously injected into nude mice (Figure 33). Clones were transiently labeled in vitro using CMTPX (red) fluorescent Cell Tracker in vitro. Optimization studies revealed that cells could be successfully labeled and maintain this dye for at least 7 days through several cell divisions (Figure 33A). Cells from most of the endothelial clones were able to home in the Matrigel plug regardless of their Tie2/VEGFR2 expression (Figure 33B). Moreover, these cells do incorporate into growing vasculature within the Matrigel plug as evident by co-staining with the endothelial marker CD31 (Figure 34). 125 DISCUSSION Differential expression of Tie2 and VEGFR2 receptors in EPC derived cells observed in our studies may potentially have important implications in the use of antiangiogenic therapies. Anti-cancer agents {e.g. bevacizumab) targeting specific endothelial receptors, such as VEGFR2 or Tie2, may not be as effective on these cells as originally thought. In the last decade, numerous research groups have isolated and successfully grown EPCs as cell colonies ex vivo from adult peripheral and umbilical cord blood MNC (Rafii and Lyden, 2003). To date, there have been no reports on isolation and identification of such cells from bovine peripheral blood. Here, we describe methodology for successful isolation of MNCs from healthy adult cows that will give rise to endothelial cell colonies in vitro. In contrast to the methodology of many research groups, the method that we employed did not involve FACS based identification of progenitors (using specific markers such as CD34, etc.) before the initial seeding of peripheral blood MNCs into tissue culture plates. Instead, our method involved seeding the entire MNC isolate from the peripheral blood of the cow, without any pre-selection, a protocol that some other research groups also utilized (Ingram et al., 2005; Ingram et al., 2004; Kalka et al., 2000a). In addition, this method is simple, does not require sophisticated equipment and is cost and time effective. It yields on average 1.8 million mononuclear cells/ml of bovine peripheral blood which translated into 4-10 colonies, appearing anywhere 4-21 days after initial seeding. This allowed us to observe the wide range of peripheral blood cell outgrowth in vitro and to study in more detail the cells that 126 can arise when subjected to specific culture conditions. We feel that this setting better represents the in vivo situation, where circulating adult EPCs home to the site of angiogenesis, and where the presence of natural matrix would support their adhesion and provide an ideal microenvironment for their differentiation. In addition, many groups have focused on CFU-ECs as the in vitro equivalent to bone marrow derived endothelial cells (Gehling et al., 2007; Ingram et al., 2004; Yoder, 2009). However, in 2007 Yoder et al. showed, using two different methods of isolation, that in fact endothelial colony forming cells (ECFC) give rise to cells that express endothelial markers while CFU-ECs give rise to cells expressing endothelial and hematopoietic markers. More importantly, ECFC but not CFU-ECs formed functional human-murine chimeric vessels when injected into mice, leading to the conclusion that ECFCs actually function as EPCs while CFU-ECs do not (Yoder et al., 2007). Based on these criteria, the late outgrowth endothelial clones we obtained and characterized in this study can be considered the bovine equivalent of these ECFCs. The methodology used in our experiments allowed us to observe the outgrowth of both CFU-EC and ECFC in MNCs from bovine blood. Consistent with other reports, we found that CFU-EC did not replate well (Yoder et al., 2007) and therefore were not the focus of further phenotype analysis. In addition, our in vivo studies confirmed that we in fact isolated ECFCs allowing us to study their differentiation, proliferation, and phenotype, specifically, their expression of Tie2 and VEGFR2 receptors. This allowed us to get a better insight into the receptor tyrosine kinases expression patterns of ECFC generated cells and to evaluate them as possible targets for antiangiogenic targeted therapies, many of which are currently being utilized as cancer therapies. 127 According to our model, our studies show that extracellular matrix does not play a significant role in differentiation of peripheral blood MNCs into endothelial cells in cattle. Endothelial clones derived from progenitor cells also had interesting and surprising surface marker and RTK expression profiles, especially with regard to Tie2 and VEGFR2. The significance of this heterogeneous expression was investigated through in vitro and in vivo studies. Although clones expressing low levels of both Tie2 and VEGFR2 were unable to form cords on Matrigel, cells with high Tie2/low VEGFR2 or the reciprocal did not display any detectable impairment in this assay. In fact, endothelial cells, in order to form cord-like structures in Matrigel, have to sprout and they do so by forming a migrating column. Cells situated at the tip of the sprout ("tip cells") sense and navigate the environment using VEGF as a major guide (Carmeliet et al., 2009; De Smet et al., 2009; Gerhardt and Betsholtz, 2005). Cells in the stalk proliferate and follow the "tip" cell, forming a lumen (Carmeliet et al., 2009). This lumen formation is carefully orchestrated by VEGF distribution and it's binding to VEGFR2 on the stalk cells (Gerhardt and Betsholtz, 2005). Thus, in our endothelial clones low in VEGFR2, this signaling may be non-efficient and therefore, cord-like structures were not produced. Interestingly, it has been reported that Tie2 is expressed in stalk cells, but not in tip cells (Yana et al., 2007). In our model, Tie2 seems to be able to rescue this non-cord forming phenotype, but the exact mechanism for this still needs to be elucidated. In contrast, there were no detectable differences in ability to home into vascularized Matrigel plugs between endothelial clones with differential expression of Tie2 and VEGFR2. It is important to note that the cells described here may have acquired or lost properties during their culture that may or may not impact their behavior in our assays. 128 Compared to their non-cultured counterpart, these in vitro derived cells may be different from their circulating counterparts in the blood, from which they originate and which are of interest to our understanding of adult vasculogenesis. In fact, this question is relevant to any in vitro defined cell population and should cause one to be careful in directly comparing the role, phenotype and function of cultured cells with respect to their original in vivo (non-passaged) precursors (Amatschek et al., 2007). These studies potentially have important implications in the use of antiangiogenic therapies. Anti-cancer agents (e.g. bevacizumab) targeting specific endothelial receptors, such as VEGFR2, are already in clinical use, some with mixed results (Hsu and Wakelee, 2009). Our results can also potentially explain the heterogeneity in expression of some endothelial receptors, such as Tie2 and VEGFR2 that we have reported in the vasculature of many human cancers (Fathers et al., 2005); (Patten et al, unpublished results). Recruiting endothelial progenitor cells with the potential for differential expression of endothelial specific receptors could hinder the effectiveness of antiangiogenic agents targeted towards that receptor. As well, our findings have implications for vascular tissue engineering and regenerative medicine, as the functional implications of these endothelial differentiation patterns remains to be fully determined. 129 B 120" 100' * Number of on. •f" "islands" observed 60" 40" 20" 0 Collagen FN Uncoated 3.00 2.50 Relative number 2 of 1.50 "islands" 1.00 0.50 0.001 3 day 5 day 7 day 7 day in hypoxia Figure 28. Phenotype of cells arising from peripheral blood MNCs. (A) Phase contrast images of cell "islands" (a) and endothelial clone (b). Scale bar is 100 fm (B) Effect of different plating conditions on colony "island" formation of bovine mononuclear cells - the number of colony "islands" counted on collagen and fibronectin (FN) coated plates was statistically significant (*p<0.05) from non-coated (control) plates. (C) The number of colony "islands" observed in non- coated plates where non-adherent cells were removed 3,5 or 7 days after incubation in normoxic conditions and 7 days after incubation in hypoxic conditions. No statistically significant difference (p>0.05) was observed. 130 NC 1 2 3 4 5 6 230 kDa - VEGFR2 160 kDa Tie2 130 kDa • • N-cadherin 55 kDa - a-tubulin B FN 1 2 3 4 5 6 7 8 9 10 11 12 230 kDa • - VEGFR2 160 kDa • # * m* -Tie2 ft 180 kDa • . VEGFR 1 130 kDa .N-cadherin 130 kDa - CD31 55 kDa - a-tubulin FN 1 2 3 4 5 230 kDa - VEGFR2 160 kDa Tie2 130 kDa • N-cadherin 55 kDa • a-tubulin Figure 29. Western blot analysis of isolated bovine clones grown on different extracellular matrices (A) A composite blot of 6 different clones grown on non- coated plates (NC) (control) shows clear expression of endothelial markers. Blot also demonstrates that some of the clones express N-cadherin. (B) A composite blot of 12 different clones from the same isolation as in (A) but grown on fibronectin (FN) or collagen (C). (C) Another composite blot of a different bovine MNC isolation showing phenotype of clones obtained from collagen and fibronectin coated plates. There was no PDGFRP or smooth-muscle actin (SMA) staining observed when these clones were tested (results not shown). 131 NC C FN 1 2 3 4 5 6 7 11 12 13 0 230 kDa • iiB • VEGFR2 160 kDa • Tie2 55 kDa • a-tubulin B hiBbTtei/iiig^^SSil? highTie2)lowVEGFR2 lowTie2/highVEGFR2 lowTie2/lowVEGFR2 Figure 30. Phenotype of clones with a differential expression of Tie2/VEGFR2 receptors. (A) Western blot analysis of representative clones (framed) isolated from MNCs grown on either collagen (C) or fibronectin (FN) matrices or non-coated plates (NC) revealed highly differential expression of VEGFR2 and Tie2; proteins were normalized to a-tubulin. Representative clones are marked with boxes - clone 6 has low expression of both Tie2/VEGFR2, a clone 8 has high VEGFR2 and low Tie2 expression,, clone 11 has high Tie2 and VEGFR2 expression and clone 13 has low VEGFR2 and high Tie2 expression. (B) Clones with high or low Tie2 and/or high and low VEGFR2 do not reveal any obvious differences in morphology by phase contrast microscopy. Scale bar is 100 /um 132 1234 5 678 9 10 230 kDa • • VEGFR2 160 kDa Tie2 130 kDa' CD31 116 kDa CD45 55 kDa- a-tubulin Figure 31. Expression of CD45 marker by isolated endothelial clones. Random clones from several isolations were stained for the expression of monocyte marker CD45, revealing that all the clones are negative for this marker. Lanes 9 and 10 depict positive controls for CD45, BJAB (T-cell lymphoma cells) and lysed buffy-coat mononuclear cells obtained from bovine peripheral blood, respectively. 133 Figure 32. Ability of clones with different expression of Tie2 and VEGFR2 receptors to form cord-like structures on Matrigel. Representative images depict that clones with low expression of Tie2 and VEGFR2 do not form cord-like structures on Matrigel while higher expression of either Tie2 or VEGFR2 seems to render them able to form these structures on Matrigel. Scale bar is 10 /jm 134 30 min 7 days CMTPX CMTPX 7 days 7 da^s DAPI Overlay x % " -« * - —-— B Figure 33. Detection of fluorescently labeled clones. (A) Optimization of cell tracking technique for fluorescently labeling cells using CellTracker Red CMTPX. As seen from fluorescent images, endothelial cells are well labeled with the tracker after 30 minutes and maintain robust fluorescence even 7 days post labeling, through multiple cell divisions. Scale bar is 10 fxm. (B) Detection of fluorescently labeled cells 7 days post tail vein injection into immunodeficient mice with established subcutaneous Matrigel plugs. Representative fluorescent images clearly showing presence of CMTPX fluorescently labeled cells in the Matrigel plug (arrows). Counterstained with DAPI (nuclear stain). Scale bar is 100 jum 135 CD31 CD31 / • CMTPX CMTPX i1 / DAPI DAPI i 1 / CD31 /CMTPX merge CD31/CMTPX merge i i / . - i Figure 34. Infiltration of endothelial cells and blood vessel formation in Matrigel plug. Cryosections from representative Matrigel plugs. Two different endothelial clones (Tie2 high/VEGFR2 low on the left and Tie2 low/VEGFR2 high on the right) were used to demonstrate the presence of endothelial cells incorporated into blood vessels in the Matrigel plug. Arrows show CD31/CMTPX dual positive cells located in the formed blood vessels of the Matrigel plug. Scale bar is 100 jum. 136 GENERAL DISCUSSION Due to the vast amount of research in the past few decades in the field of tumor angiogenesis and tumor progression, it is now known that mutations of oncogenes and tumor suppressor genes can often lead to transformation of a normal cell to become a malignant cell (Lehman et al., 1991; Rak et al., 1995; Suarez, 1989). However it is also widely known that most malignant cells cannot grow to a clinically detectable tumor mass in the absence of blood vessels (Folkman, 1971; Hillen and Griffioen, 2007). Thus, a clinically manifested large tumor has to switch on an angiogenic phenotype to support its growth. The switch of an angiogenic phenotype may represent an imbalanced expression of angiogenic factors and angiogenic inhibitors (Bergers and Benjamin, 2003). Overexpression of angiogenic factors and down-regulation of angiogenesis inhibitors are both necessary and sufficient to induce new blood vessels growth, and these two processes usually occur simultaneously to switch on tumor angiogenesis (Bergers and Benjamin, 2003). We examined two of these opposing molecules, pro-angiogenic VEGF and antiangiogenic TSP-1 in this thesis in the context of malignant melanoma, and confirmed inverse expression of these factors in vitro as had been previously reported in the literature (Greenaway et al., 2007; Straume and Akslen, 2001). In order to determine the exact, if any, relationship between VEGF and TSP-1, in vivo studies should be designed to look at this in more detail. Besides pro-angiogenic and antiangiogenic molecules, this thesis dealt with examination of two important tyrosine kinase receptors, Tie2 and VEGFR2. In Chapter 2, we investigated the role of VEGFR2 receptor in cancer; in particular we identified a 137 functional VEGFR2 receptor in primary and metastatic melanoma cell lines, isolated from the same patient. Having these two host-matched cell lines, allowed us to study the role of VEGF, VEGFR2 and TSP-1 in tumor growth and how they might change with progression. In vitro studies revealed that while VEGF is significantly upregulated between primary and metastatic melanoma cells, TSP-1 gets significantly downregulated, and the levels of VEGFR2 decreased. In fact, previous reports show malignant progression in melanoma is associated with reduced TSP expression and increased VEGF expression (Fontanini et al., 1997b; Gorski et al., 2003; Straume and Akslen, 2001; Zabrenetzky et al., 1994). To my knowledge, there are no published studies that evaluated changes in the expression of VEGFR2 on cancer cells during tumor progression. When we used the anti-VEGF therapy bevacizumab, currently in clinical use, we expected to target not only endothelial cells in our xenografted melanomas, but also cancer cells that express VEGFR2 receptor. Interestingly, treatment of both primary and metastatic melanoma xenografts did not result in decreased tumor growth, as expected, despite significant antiangiogenic effects. One of the main reasons for this may be differences in reliance on VEGF signaling in melanoma cells (Yu et al., 2001). In fact, a recent study suggested that targeting EGFR on endothelial cells of melanoma xenografts using gefitinib, allowed tumors to switch from dependence on EGFR activity to signaling with VEGFR2 (Amin et al., 2008). Although not reported, the opposite could also be happening. When Dr. Judah Folkman proposed, about 40 years ago, that tumors cannot grow and metastasize without blood vessels (Folkman, 1971) a new wave of research was guided towards the inhibition of various endothelial pathways that would cause endothelial cell apoptosis to occur. 138 Many of these inhibitors showed only moderate results with great variations, even between different tumor growth sites in the same patient (Jung et al., 2000; McCarthy, 2001). In fact, it has been shown that increased malignancy is associated with a decrease in vascular dependence of tumor cells related to blood vessel proximity of these cancer cells (Rak et al., 1996; Yu et al., 2001). "Distal" cells isolated from hypoxic regions in melanoma, were found to form more aggressive tumors in mice than melanoma cells isolated from areas "proximal" to blood vessels. Interestingly, growth of these more aggressive tumor cells was associated with lower blood vessel density compared to the growth of less aggressive tumor cells that were associated with higher blood vessel density (Yu et al., 2001). Therefore, what might have happened in our bevacizumab treated tumors may be due to the vascular dependence of the cells - the more aggressive these cells became, the less they relied on their blood vessels to supply oxygen and nutrients for survival allowing them to continue proliferating. This translated into non- observable anti-tumor effect despite considerable antiangiogenic effect. Decrease in vascular dependence may be due to the ability of cancer cells to adapt to tumor microenvironmental conditions, which they are able to do through the acquisition of various mutations in their genome giving them the ability to survive harsh conditions and avoid apoptosis. This advantage arises from oncogenic genetic changes, such as loss or mutation of the p53 tumor suppressor gene that gives cancer cells ability to survive in hypoxic culture conditions better than the cells that poses wild-type p53 (Yu et al., 2002). In addition to p53, activation of oncogenes such as ras, src and HER2 may also give cells this advantage (Rak et al., 1995). Although WM115 and WM239 139 melanoma cell lines have previously been characterized as p53 wild-type, mutation of other oncogenes and tumor suppressor genes have been less characterized (Smalley et al., 2007). In fact, the RAS/RAF/MEK/ERK signaling pathway has emerged as a major player in the induction and maintenance of melanoma, particularly the protein kinase BRAF, mutated in approximately 44% of melanoma cases including our cell lines WM115 and WM239 (Dhomen and Marais, 2009; Grbovic et al., 2006). Currently, BRAF inhibitors are being investigated in the clinical setting (Dhomen and Marais, 2009). In Chapter 3, Tie2 expression was examined in tumor endothelium of melanoma xenografts in the context of Tie2 targeted and antiangiogenic non-targeted LDM CTX therapy. Although the combination of such therapies seems in principle to be effective and is used currently in numerous clinical and pre-clinical trials (Brown et al., 2010; Daenen et al., 2009; Dellapasqua et al., 2008; Hurwitz et al, 2004), our data show that not all combinations of therapies may translate into decreased tumor growth and tumor burden. In particular, as seen in our studies the Tie2 inhibitor TekdeltaFc combined with LDM CTX had adverse effects on tumor growth of melanoma xenografts. Tumor growth was improved when the two therapies were combined, compared to when either was administered alone. This was not expected since TekdeltaFc and LDM CTX presumably target two different pathways - TekdeltaFc targets Tie2 and angiopoietins (Das et al., 2003), while LDM CTX has cytotoxic effects on endothelial cells in the tumors and was proposed to decrease the production of VEGF by increasing the production of Thrombospondin-1 (Hamano et al., 2004). Interestingly, when investigated in vitro, a similar effect is observed. Individually both therapies decreased phosphorylation of 140 endothelial Tie2 receptor 24 hours after administration but when combined, the phosphorylation of Tie2 receptor levels was enhanced compared to controls. This indicated that the inhibitory activity of TekdeltaFc on Tie2 receptor and angiopoietins is somehow modulated by cyclophosphamide administration. We also noticed significantly increased VEGF expression in combined tumors suggesting the interaction of Tie2 and VEGF signaling pathways and a possibility of VEGF indirectly activating Tie2 receptor, a notion that has been previously reported (Singh et al., 2009). But thus far, the exact mechanism of this interaction still needs to be investigated in more detail. Looking at the current experimental evidence, there are at least four distinctive adaptive mechanisms of resistance to antiangiogenic therapies, each of which could explain the possible results we encountered in pre-clinical trials described in this thesis. The first is the activation/upregulation of alternative pro-angiogenic signaling pathways in the tumors; second, recruitment of bone-marrow derived pro-angiogenic cells; third, increased pericyte coverage of the tumor vasculature leading to more stable vessels refractory to degeneration, and fourth, activation and enhancement of invasion and metastasis providing access to normal tissue vasculature (Bergers and Hanahan, 2008). Upregulation of pro-angiogenic molecules could have occurred in both of our trials, bevacizumab treated as well as TekdeltaFc/LDM CTX trial. Such up-regulation has been reported in the literature before, using preclinical models of Ripl-Tag2 mice that were treated with monoclonal antibody (DC101) against VEGFR2 signaling (Casanovas et al., 2005). In this experiment, initial response (tumor stasis) was noted but was followed by tumor regrowth. These relapsing tumors were found to expresses higher levels of other pro-angiogenic factors, such as FGF. We observed this in our 141 TekdeltaFc/LDM CTX trial where initially there were anti-tumor effects. Once the initial anti-tumor effects subsided, these melanoma xenografts all started growing at the same rate, regardless of treatment. VEGF ELISA confirmed that there was significant production of the pro-angiogenic molecule VEGF compared to untreated tumors. We did not observe this in the tumors from our bevacizumab trial, but these tumors may have upregulated a different pro-angiogenic molecule such as FGF (as seen in Casanovas et al., 2005), to avoid the VEGF-blockade. In addition, Zaghloul et al. reported that VEGF- blockade rapidly elicits alternative proangiogenic pathways in neuroblastoma, such as upregulation of placental growth factor (Zaghloul et al., 2009). More studies need to be performed to determine the exact mechanism of the antiangiogenic resistance apparently occurring in these trials. In addition, upregulation of pro-angiogenic factors can also lead to the recruitment of various bone-marrow derived cells, such as vascular (both endothelial and mural cell/pericyte) progenitor cells and vascular modulatory cells, which together have the ability to form new blood vessels in tumors (Asahara et al., 1997; Asahara et al., 1999). While endothelial and pericyte progenitors can differentiate into endothelial and pericyte cells in the tumors, pro-angiogenic monocytes such as tumor-associated macrophages (Pollard, 2004) and immature Tie2-expressing monocytes (De Palma et al., 2007), VEGFR 1 positive hemangiocytes (Kaplan et al., 2005) and CDllb positive myeloid cells (Yang et al., 2004) are also thought to contribute to tumor angiogenesis. These may act as vascular modulators expressing cytokines and growth factors but themselves may not participate directly in the formation of the vasculature (Du et al., 2008). Tumor-associated macrophages (TAM) play an important role in tumorigenesis. 142 Derived from circulating monocytes (which differentiate into macrophages upon homing to tumors) these cells may convert to a protumoral phenotype once influenced by the tumor microenvironment (Coffelt et al., 2009; Solinas et al., 2009). Moreover, recently Venneri et al. identified a subset of Tie2- expressing monocytes (TEM) located at the sites of angiogenesis in tumours. These have now been implicated in human cancer angiogenesis and progression and may represent previously un-recognized targets of anti- cancer therapies (De Palma et al., 2007; Venneri et al., 2007), since selective depletion of these Tie2-expressing monocytes in tumor-bearing mice inhibited tumor angiogenesis and growth. TEMs are also currently being evaluated as cellular vehicles for gene delivery to tumors (De Palma et al, 2007). We did not find expression of the hematopoietic CD45 marker in our endothelial clones, pointing to the fact that the clones we isolated are unlikely derived from Tie2-expressing monocytes or similar cells, since these, according to De Palma et al. express CD45 marker (De Palma et al., 2007; De Palma and Naldini, 2006). Another possible reason for our observed decrease in blood vessel density without decreased tumor volume may be due to vessel normalization induced in response to antiangiogenic therapy. In fact, Dr. Rakesh Jain proposed initially that antiangiogenic therapy will transiently improve both the structure and the function of tumor vessels. However, sustained or aggressive antiangiogenic therapy may in fact eventually prune away these functional vessels, resulting in vasculature resistant to further treatment and inadequate delivery of drugs or oxygen to the tumor (Jain, 2001). Increased and tighter pericyte coverage that protects tumor blood vessels has been specifically observed upon inhibition of VEGF signaling, leading to the reduction of blood vessel density, and 143 leaving functional vessels that remain tightly covered with pericytes (Willett et al., 2004). This hypothesis is supported by the findings that tumor vessels lacking adequate pericyte coverage are more vulnerable to VEGF inhibition and that pericytes may express enough VEGF and potential other growth factors to support endothelial survival (Darland et al., 2003; Erber et al., 2004; Greenberg et al., 2008; Hoffmann et al., 2005). Specifically, bevacizumab alone is able to decrease blood vessel density in both human cancer and mouse tumor models by 30-50% after two weeks of treatment (Willett et al., 2004). Besides microvascular density, vascular volume and interstitial blood pressure were also reduced but without a change in the uptake of nutrients by these "normalized" vessels - showing a more efficient delivery of chemotherapeutic agents to tumors than before bevacizumab treatment (Willett et al., 2004). Microvasculature of different organs is phenotypically distinct from one another due to the specific microenvironment that can affect gene expression in tumors growing at these different sites (Auerbach, 1991; Gutman et al., 1995; Liu et al., 2001). In addition, cancer cells themselves can alter endothelial phenotype making it challenging for antiangiogenic therapies, which can produce the desired antiangiogenic effect at one organ site but not another (Auerbach, 1991; Eberhard et al., 2000). Vasculature of tumors differs from that of normal tissues in respect to pericyte coverage, abnormal branching patterns and absence of continuous endothelial lining (Hashizume et al., 2000; Morikawa et al., 2002). Endothelial cells in tumours have biochemical and functional characteristics absent from endothelial cells in normal vascular beds. For instance, quiescence of endothelial cells is maintained through the products expressed by pericytes, such as angiopoietin-1 and transforming growth factor - P, etc. (Fukuhara et al., 2010; Nishishita 144 and Lin, 2004; Papapetropoulos et al., 1999; Sato and Rifkin, 1989). Endothelial phenotype can also be affected by the factors in tumor microenvironment such as growth rate of tumor cells, angiogenic stimulus and nature of the host cells (Jung et al., 2002). Endothelial cells in the tumor microenvironment interact not only with cancer cells but also with immune cells, fibroblasts, pericytes and extracellular matrix, which all can have influence on endothelial phenotype (Jung et al., 2002). The physical relationship and cross-talk of these cells may determine gene expression, phenotype and behaviors of both cancer and endothelial cells. When designing antiangiogenic therapies, all these should be taken into account (Brown and Giaccia, 1998). One of the most common problems encountered in clinical trials with antiangiogenic drugs targeting a particular receptor, for instance Tie2, or angiogenic factor, such as VEGF, is that in many cases it is unclear whether a patient's cancer expresses the target of the drug or if this interaction between the drug and the target can stop cancer growth. Many of these drugs are used in combination which translates into large financial burden on health care systems around the world, and stimulated the search for a "surrogate" marker for determining the optimal dose of these drugs (Bertolini et al., 2006). As we described earlier, combining antiangiogenic therapies does have its validity although, as we show in our studies, may also have unexpected results. Some of the more common surrogate markers for the effectiveness of antiangiogenic drugs are vascular density measurements (Hlatky et al., 2002) and measurements of circulating levels of angiogenic growth factors such as VEGF, bFGF, HGF, etc. (Bertolini et al., 2006; Motzer et al., 2006a; Motzer et al., 2006b). It was noticed in 1978 that cells of endothelial origin are circulating in the blood and were found to be increased in many pathological 145 conditions, including cancer (Hladovec, 1978). Some of these cells displayed mature endothelial characteristics (fully differentiated endothelial cells) while others expressed markers of stem-like or progenitor-like phenotype with an ability to home to the sites of angiogenesis (Blann and Pretorius, 2006; Blann et al., 2005). Many have tried to use these circulating endothelial/progenitor cells as a surrogate for the effectiveness of antiangiogenic drugs (Mancuso et al., 2003; Martin-Padura and Bertolini, 2009; Twardowski et al., 2008). As previously discussed, there are numerous markers used by various groups used to identify the hierarchy of these endothelial progenitor cells and their level of differentiation. Tie2 and VEGFR2 levels are reported to be expressed in circulating endothelial cells and their levels are reported to increase with increased maturation (Bompais et al., 2004). We reported in our studies differential expression of Tie2 and VEGFR2 receptors of these isolated cells which may make it difficult to use them as a "surrogate" marker for antiangiogenic drugs, and may interfere with their usefulness as target for antiangiogenic agents for these particular receptors. Although we show that both of these endothelial phenotypes, ones expressing high Tie2 and low VEGFR2 as well as high VEGFR2 and low Tie2, can home in our Matrigel plugs, it would be interesting to know if these cells changed or maintained their expression patterns of these receptors once incorporated into Matrigel microcirculation. Future studies are required to answer this question. 146 SUMMARY AND CONCLUSIONS The research described in this thesis dealt with in-depth examination of two critical tyrosine kinase receptors, Tie2 and VEGFR2 that play major roles in formation and stability of blood vessels during the process of angiogenesis. Targeted (Tie2 inhibitor TekdeltaFc and anti-VEGF antibody, bevacizumab/Avastin) and non-targeted (LDM CTX) antiangiogenic therapies were employed to inhibit these pathways, to obtain insight into the functionality in tumor vasculature. We noticed that when administered alone, TekdeltaFc and LDM CTX induced only transient anti-tumor properties, but when combined, these therapies appear to "cancel" each other, suggesting a complex interaction of these pathways. Here, we also report the expression of VEGFR2 receptor by melanoma cell lines WM115 and WM239, and its contribution to both autocrine and intracrine VEGF/VEGFR2 signaling. Besides signaling survival pathways as reported in other studies, we suggest that VEGFR2 may be primarily to aid in migration and adhesion of melanoma cells, as seen through the decreased phosphorylation of focal adhesion kinase when cells were exposed to the VEGFR2 small-molecule inhibitor ZM323881. In fact, our studies suggest that small molecule inhibitors, such as ZM323881, may be more potent inhibitors of VEGFR2 signaling than bevacizumab. Bevacizumab had an ability to produce antiangiogenic effects on melanoma xenografts, but also produced a sustained increase in phosphorylation of VEGFR2 receptor in vitro, the mechanisms of which still need to be elucidated. Finally, bone-marrow derived circulating endothelial progenitor cells were isolated from bovine blood, differentiated in vitro into endothelial cells and characterized with regards to their differential expression of Tie2 and VEGFR2. We also showed that 147 these cells have an ability to home to vasculature in Matrigel plugs when injected into immune deficient mice. All of these studies gave us a better understanding of the expression of Tie2 and VEGFR2 receptors in endothelial cells, but also in circulating endothelial progenitor derived cells with variable phenotype and cancer cells themselves. Together, these findings expand out appreciation of the complexity of angiogenic receptor expression in tumors and its potential impact on progression and response to therapy. 148 REFERENCES • Abedi, H., and Zachary, I. (1997). Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. J Biol Chem 272, 15442-15451. • Abranis, T. J., Lee, L. B., Murray, L. J., Pryer, N. K., and Cherrington, J. M. (2003a). SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer. Mol Cancer Ther 2, 471-478. • Abrams, T. J., Murray, L. J., Pesenti, E., Holway, V. W., Colombo, T., Lee, L. B., Cherrington, J. M., and Pryer, N. K. (2003b). Preclinical evaluation of the tyrosine kinase inhibitor SU11248 as a single agent and in combination with "standard of care" therapeutic agents for the treatment of breast cancer. Mol Cancer Ther 2, 1011-1021. • Abramsson, A., Lindblom, P., and Betsholtz, C. (2003). Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors. J Clin Invest 112, 1142-1151. • Adams, J. M., and Cory, S. (2001). Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 26, 61-66. • Aesoy, R., Sanchez, B. C., Norum, J. H., Lewensohn, R., Viktorsson, K., and Linderholm, B. (2008). An autocrine VEGF/VEGFR2 and p38 signaling loop confers resistance to 4-hydroxytamoxifen in MCF-7 breast cancer cells. Mol Cancer Res 6, 1630-1638. • Albo, D., Granick, M. S., Jhala, N., Atkinson, B., and Solomon, M. P. (1994). The relationship of angiogenesis to biological activity in human squamous cell carcinomas of the head and neck. Ann Plast Surg 32, 588-594. • Amatschek, S., Kriehuber, E., Bauer, W., Reininger, B., Meraner, P., Wolpl, A., Schweifer, N., Haslinger, C., Stingl, G., and Maurer, D. (2007). Blood and lymphatic endothelial cell-specific differentiation programs are stringently controlled by the tissue environment. Blood 109, 4777-4785. • Amin, D. N., Bielenberg, D. R., Lifshits, E., Heymach, J. V., and Klagsbrun, M. (2008). Targeting EGFR activity in blood vessels is sufficient to inhibit tumor growth and is accompanied by an increase in VEGFR-2 dependence in tumor endothelial cells. Microvasc Res 76, 15-22. • Apelgren, L. D., and Bumol, T. F. (1989). Biosynthesis and secretion of thrombospondin in human melanoma cells. Cell Biol Int Rep 13, 189-195. • Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der Zee, R., Li, T., Witzenbichler, B., Schatteman, G., and Isner, J. M. (1997). Isolation of putative progenitor endothelial cells for angiogenesis. Science 275, 964-967. • Asahara, T., Takahashi, T., Masuda, H., Kalka, C., Chen, D., Iwaguro, H., Inai, Y., Silver, M., and Isner, J. M. (1999). VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 18, 3964-3972. • Atkins, M., Jones, C. A., and Kirkpatrick, P. (2006). Sunitinib maleate. Nat Rev Drug Discov 5, 279-280. 149 Auerbach, R. (1991). Vascular endothelial cell differentiation: organ-specificity and selective affinities as the basis for developing anti-cancer strategies. Int J Radiat Biol 60, 1-10. Bachelder RE, Crago A, Chung J, Wendt MA, Shaw LM. (2001) Vascular endothelial growth factor is an autocrine survival factor for neurophilin- expressing breast carcinoma cells. Cancer Res 61: 5736-5740. Backer, M. V., Hamby, C. V., and Backer, J. M. (2009). Inhibition of vascular endothelial growth factor receptor signaling in angiogenic tumor vasculature. Adv Genet 67, 1-27. Baish, J. W., and Jain, R. K. (2000). Fractals and cancer. Cancer Res 60, 3683- 3688. Barleon, B., Sozzani, S., Zhou, D., Weich, H. A., Mantovani, A., and Marme, D. (1996). Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 87, 3336-3343. Barwick, T„ Bencherif, B., Mountz, J. M., and Avril, N. (2009). Molecular PET and PET/CT imaging of tumour cell proliferation using F-18 fluoro-L-thymidine: a comprehensive evaluation. Nucl Med Commun 30, 908-917. Bates, D. O., and Harper, S. J. (2002). Regulation of vascular permeability by vascular endothelial growth factors. Vascul Pharmacol 39, 225-237. Bellamy, W. T., Richter, L., Frutiger, Y., and Grogan, T. M. (1999). Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies. Cancer Res 59, 728-733. Bergers, G., and Benjamin, L. E. (2003). Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3, 401-410. Bergers, G., and Hanahan, D. (2008). Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer 8, 592-603. Berns, E. M., Klijn, J. G., Look, M. P., Grebenchtchikov, N., Vossen, R., Peters, H., Geurts-Moespot, A., Portengen, H., van Staveren, I. L., Meijer-van Gelder, M. E., et al. (2003). Combined vascular endothelial growth factor and TP53 status predicts poor response to tamoxifen therapy in estrogen receptor-positive advanced breast cancer. Clin Cancer Res 9, 1253-1258. Bertolini, F., Paul, S., Mancuso, P., Monestiroli, S., Gobbi, A., Shaked, Y., and Kerbel, R. S. (2003). Maximum tolerable dose and low-dose metronomic chemotherapy have opposite effects on the mobilization and viability of circulating endothelial progenitor cells. Cancer Res 63, 4342-4346. Bertolini, F., Shaked, Y., Mancuso, P., and Kerbel, R. S. (2006). The multifaceted circulating endothelial cell in cancer: towards marker and target identification. Nat Rev Cancer 6, 835-845. Betsholtz, C. (2004). Insight into the physiological functions of PDGF through genetic studies in mice. Cytokine Growth Factor Rev 15, 215-228. Bittner, M., Meltzer, P., Chen, Y., Jiang, Y., Seftor, E., Hendrix, M., Radmacher, M., Simon, R., Yakhini, Z., Ben-Dor, A., et al. (2000). Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 406, 536-540. Blann, A. D., and Pretorius, A. (2006). Circulating endothelial cells and endothelial progenitor cells: two sides of the same coin, or two different coins? Atherosclerosis 188, 12-18. 150 • Blann, A. D., Woywodt, A., Bertolini, F., Bull, T. M., Buyon, J. P., Clancy, R. M., Haubitz, M., Hebbel, R. P., Lip, G. Y., Mancuso, P., et al. (2005). Circulating endothelial cells. Biomarker of vascular disease. Thromb Haemost 93, 228-235. • Blazquez C, Cook N, Micklem K, Harris AL, Gatter KC, Pezzella F. (2006) Phosphorylated KDR can be located in the nucleus of neoplastic cells. Cell Res. Jan;16(l):93-8. • Bocci, G., Francia, G., Man, S., Lawler, J., and Kerbel, R. S. (2003). Thrombospondin 1, a mediator of the antiangiogenic effects of low-dose metronomic chemotherapy. Proc Natl Acad SciU S A 100, 12917-12922. • Bogdanovic, E., Coombs, N., and Dumont, D. J. (2009). Oligomerized Tie2 localizes to clathrin-coated pits in response to angiopoietin-1. Histochem Cell Biol 132, 225-237. • Bogdanovic, E., Nguyen, V. P., and Dumont, D. J. (2006). Activation of Tie2 by angiopoietin-1 and angiopoietin-2 results in their release and receptor internalization. J Cell Sci 119, 3551-3560. • Bompais, H., Chagraoui, J., Canron, X., Crisan, M., Liu, X. H., Anjo, A., Tolla- Le Port, C., Leboeuf, M., Charbord, P., Bikfalvi, A., and Uzan, G. (2004). Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells. Blood 103, 2577- 2584. • Bond, M., Hoyle, M., Moxham, T., Napier, M., and Anderson, R. (2009). Sunitinib for the treatment of gastrointestinal stromal tumours: a critique of the submission from Pfizer. Health Technol Assess 13 Suppl 2, 69-74. • Boocock, C. A., Charnock-Jones, D. S., Sharkey, A. M., McLaren, J., Barker, P. J., Wright, K. A., Twentyman, P. R., and Smith, S. K. (1995). Expression of vascular endothelial growth factor and its receptors fit and KDR in ovarian carcinoma. J Natl Cancer Inst 87, 506-516. • Bosari, S., Lee, A. K., DeLellis, R. A., Wiley, B. D., Heatley, G. J., and Silverman, M. L. (1992). Microvessel quantitation and prognosis in invasive breast carcinoma. Hum Pathol 23, 755-761. • Breier, G. (2000). Angiogenesis in embryonic development—a review. Placenta 21 Suppl A, SI 1-15. • Brkovic, A., Pelletier, M., Girard, D., and Sirois, M. G. (2007). Angiopoietin chemotactic activities on neutrophils are regulated by PI-3K activation. J Leukoc Biol 81, 1093-1101. • Browder, T„ Butterfield, C. E., Kraling, B. M., Shi, B., Marshall, B., O'Reilly, M. S., and Folkman, J. (2000). Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60, 1878-1886. • Brown, J. L., Cao, Z. A., Pinzon-Ortiz, M., Kendrew, J., Reimer, C., Wen, S., Zhou, J. Q., Tabrizi, M., Emery, S., McDermott, B., et al. A human monoclonal anti-ANG2 antibody leads to broad antitumor activity in combination with VEGF inhibitors and chemotherapy agents in preclinical models. Mol Cancer Ther 9, 145-156. • Brown, J. L., Cao, Z. A., Pinzon-Ortiz, M., Kendrew, J., Reimer, C, Wen, S., Zhou, J. Q., Tabrizi, M., Emery, S., McDermott, B„ et al. (2010). A human monoclonal anti-ANG2 antibody leads to broad antitumor activity in combination 151 with VEGF inhibitors and chemotherapy agents in preclinical models. Mol Cancer Ther 9, 145-156. Brown, J. M., and Giaccia, A. J. (1998). The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 58, 1408-1416. Brown, L. F., Harrist, T. J., Yeo, K. T., Stahle-Backdahl, M., Jackman, R. W., Berse, B., Tognazzi, K., Dvorak, H. F., and Detmar, M. (1995). Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme. J Invest Dermatol 104, 744-749. Brown, L. F., Yeo, K. T., Berse, B., Yeo, T. K., Senger, D. R., Dvorak, H. F., and van de Water, L. (1992). Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 176, 1375-1379. Browning, A. C., Dua, H. S., and Amoaku, W. M. (2008). The effects of growth factors on the proliferation and in vitro angiogenesis of human macular inner choroidal endothelial cells. Br J Ophthalmol 92, 1003-1008. Bunone, G., Vigneri, P., Mariani, L., Buto, S., Collini, P., Pilotti, S., Pierotti, M. A., and Bongarzone, I. (1999). Expression of angiogenesis stimulators and inhibitors in human thyroid tumors and correlation with clinical pathological features. Am J Pathol 155, 1967-1976. Cabebe, E., and Wakelee, H. (2006). Sunitinib: a newly approved small-molecule inhibitor of angiogenesis. Drugs Today (Bare) 42, 387-398. Caldwell, R. B., Bartoli, M., Behzadian, M. A., El-Remessy, A. E., Al- Shabrawey, M., Piatt, D. H., and Caldwell, R. W. (2003). Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives. Diabetes Metab Res Rev 19, 442-455. Carlson, C. B., Lawler, J., and Mosher, D. F. (2008). Structures of thrombospondins. Cell Mol Life Sci 65, 672-686. Carmeliet, P., De Smet, F., Loges, S., and Mazzone, M. (2009). Branching morphogenesis and antiangiogenesis candidates: tip cells lead the way. Nat Rev Clin Oncol 6,315-326. Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K„ Eberhardt, C., et al. (1996). Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435-439. Carmeliet, P., and Jain, R. K. (2000). Angiogenesis in cancer and other diseases. Nature 407, 249-257. Casanovas, O., Hicklin, D. J., Bergers, G., and Hanahan, D. (2005). Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 8, 299-309. Chandrasekaran, L., He, C. Z., Al-Barazi, H., Krutzsch, H. C., Iruela-Arispe, M. L., and Roberts, D. D. (2000). Cell contact-dependent activation of alpha3betal integrin modulates endothelial cell responses to thrombospondin-1. Mol Biol Cell 77,2885-2900. 152 Chatzizacharias, N. A., Kouraklis, G. P., and Theocharis, S. E. (2007). Focal adhesion kinase: a promising target for anticancer therapy. Expert Opin Ther Targets 11, 1315-1328. Chen, H., Herndon, M. E., and Lawler, J. (2000). The cell biology of thrombospondin-1. Matrix Biol 19, 597-614. Chen, W. S„ Kitson, R. P., and Goldfarb, R. H. (2002). Modulation of human NK cell lines by vascular endothelial growth factor and receptor VEGFR-1 (FLT-1). In Vivo 16, 439-445. Chung, Y. C„ Hou, Y. C, Chang, C. N., and Hseu, T. H. (2006). Expression and prognostic significance of angiopoietin in colorectal carcinoma. J Surg Oncol 94, 631-638. Coffelt, S. B., Hughes, R„ and Lewis, C. E. (2009). Tumor-associated macrophages: effectors of angiogenesis and tumor progression. Biochim Biophys Acta 1796, 11-18. Coggins, P. R., Ravdin, R. G., and Eisman, S. H. (1960). Clinical evaluation of a new alkylating agent: Cytoxan (cyclophosphamide). Cancer 13, 1254-1260. Cohen, M. H., Gootenberg, J., Keegan, P., and Pazdur, R. (2007). FDA drug approval summary: bevacizumab (Avastin) plus Carboplatin and Paclitaxel as first-line treatment of advanced/metastatic recurrent nonsquamous non-small cell lung cancer. Oncologist 12, 713-718. Colleoni, M., Orlando, L., Sanna, G., Rocca, A., Maisonneuve, P., Peruzzotti, G., Ghisini, R., Sandri, M. T., Zorzino, L., Nole, F., et al. (2006). Metronomic low- dose oral cyclophosphamide and methotrexate plus or minus thalidomide in metastatic breast cancer: antitumor activity and biological effects. Ann Oncol 17, 232-238. Coomber, B. L. (1995). Suramin inhibits C6 glioma-induced angiogenesis in vitro. J Cell Biochem 58, 199-207. Costa, R., Carneiro, A., Rocha, A., Pirraco, A., Falcao, M., Vasques, L., and Soares, R. (2009). Bevacizumab and ranibizumab on microvascular endothelial cells: A comparative study. J Cell Biochem 108, 1410-1417. Coussens, L. M., Raymond, W. W., Bergers, G., Laig-Webster, M., Behrendtsen, O., Werb, Z., Caughey, G. H., and Hanahan, D. (1999). Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis. Genes Dev 13, 1382-1397. Crowley-Weber, C. L., Payne, C. M., Gleason-Guzman, M., Watts, G. S., Futscher, B., Waltmire, C. N., Crowley, C., Dvorakova, K., Bernstein, C., Craven, M., et al. (2002). Development and molecular characterization of HCT-116 cell lines resistant to the tumor promoter and multiple stress-inducer, deoxycholate. Carcinogenesis 23, 2063-2080. Daenen, L. G., Shaked, Y., Man, S., Xu, P., Voest, E. E., Hoffinan, R. M., Chaplin, D. J., and Kerbel, R. S. (2009). Low-dose metronomic cyclophosphamide combined with vascular disrupting therapy induces potent antitumor activity in preclinical human tumor xenograft models. Mol Cancer Ther 8, 2872-2881. 153 Dameron, K. M., Volpert, O. V., Tainsky, M. A., and Bouck, N. (1994). Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265, 1582-1584. Darland, D. C., Massingham, L. J., Smith, S. R., Piek, E., Saint-Geniez, M., and D'Amore, P. A. (2003). Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Dev Biol 264, 275-288. Das, A., Fanslow, W., Cerretti, D., Warren, E., Talarico, N., and McGuire, P. (2003). Angiopoietin/Tek interactions regulate mmp-9 expression and retinal neovascularization. Lab Invest 83, 1637-1645. Davidoff, A. M„ Ng, C. Y., Brown, P., Leary, M. A., Spurbeck, W. W., Zhou, J., Horwitz, E., Vanin, E. F., and Nienhuis, A. W. (2001). Bone marrow-derived cells contribute to tumor neovasculature and, when modified to express an angiogenesis inhibitor, can restrict tumor growth in mice. Clin Cancer Res 7, 2870-2879. Davis, S., Papadopoulos, N., Aldrich, T. H., Maisonpierre, P. C, Huang, T., Kovac, L., Xu, A., Leidich, R., Radziejewska, E., Rafique, A., et al. (2003). Angiopoietins have distinct modular domains essential for receptor binding, dimerization and superclustering. Nat Struct Biol 10, 38-44. Dawson, D. W., Pearce, S. F., Zhong, R., Silverstein, R. L., Frazier, W. A., and Bouck, N. P. (1997). CD36 mediates the In vitro inhibitory effects of thrombospondin-1 on endothelial cells. J Cell Biol 138, 707-717. de Castro Junior, G., Puglisi, F., de Azambuja, E., El Saghir, N. S., and Awada, A. (2006). Angiogenesis and cancer: A cross-talk between basic science and clinical trials (the "do ut des" paradigm). Crit Rev Oncol Hematol 59, 40-50. de Fraipont, F„ Nicholson, A. C., Feige, J. J., and Van Meir, E. G. (2001). Thrombospondins and tumor angiogenesis. Trends Mol Med 7, 401-407. de Jong, J. S„ van Diest, P. J., van der Valk, P., and Baak, J. P. (1998). Expression of growth factors, growth inhibiting factors, and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops. J Pathol 184, 44-52. De Palma, M., Murdoch, C., Venneri, M. A., Naldini, L., and Lewis, C. E. (2007). Tie2-expressing monocytes: regulation of tumor angiogenesis and therapeutic implications. Trends Immunol 28, 519-524. De Palma, M., and Naldini, L. (2006). Role of haematopoietic cells and endothelial progenitors in tumour angiogenesis. Biochim Biophys Acta 1766, 159-166. De Smet, F., Segura, I., De Bock, K., Hohensinner, P. J., and Carmeliet, P. (2009). Mechanisms of vessel branching: filopodia on endothelial tip cells lead the way. Arterioscler Thromb Vase Biol 29, 639-649. de Vries, C., Escobedo, J. A., Ueno, H., Houck, K., Ferrara, N., and Williams, L. T. (1992). The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 255, 989-991. Decaussin, M., Sartelet, H., Robert, C., Moro, D., Claraz, C., Brambilla, C., and Brambilla, E. (1999). Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-Rl-Fltl and VEGF-R2-Flk 1 /KDR) in non-small cell 154 lung carcinomas (NSCLCs): correlation with angiogenesis and survival. J Pathol 188, 369-377. Dellapasqua, S., Bertolini, F., Bagnardi, V., Campagnoli, E., Scarano, E., Torrisi, R„ Shaked, Y„ Mancuso, P., Goldhirsch, A., Rocca, A., et al (2008). Metronomic cyclophosphamide and capecitabine combined with bevacizumab in advanced breast cancer. J Clin Oncol 26, 4899-4905. Dellinger, M., Hunter, R., Bernas, M., Gale, N., Yancopoulos, G., Erickson, R., and Witte, M. (2008). Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice. Dev Biol 319, 309-320. Denhardt, D. T. (1996). Oncogene-initiated aberrant signaling engenders the metastatic phenotype: synergistic transcription factor interactions are targets for cancer therapy. Crit Rev Oncog 7, 261-291. Detmar, M., Brown, L. F., Berse, B., Jackman, R. W., Elicker, B. M., Dvorak, H. F., and Claffey, K. P. (1997). Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin. J Invest Dermatol 108, 263-268. Detmar, M., Brown, L. F., Claffey, K. P., Yeo, K. T„ Kocher, O., Jackman, R. W., Berse, B., and Dvorak, H. F. (1994). Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med 180, 1141-1146. Dhomen, N., and Marais, R. (2009). BRAF signaling and targeted therapies in melanoma. Hematol Oncol Clin North Am 23, 529-545, ix. Dias, S., Hattori, K., Heissig, B., Zhu, Z., Wu, Y., Witte, L., Hicklin, D. J., Tateno, M., Bohlen, P., Moore, M. A., and Rafii, S. (2001). Inhibition of both paracrine and autocrine VEGF/ VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias. Proc Natl Acad Sci U S A 98, 10857-10862. Dittmann, K., Mayer, C„ and Rodemann, H. P. (2010). Nuclear EGFR as novel therapeutic target: insights into nuclear translocation and function. Strahlenther OnkoliSd, 1-6. Dougher, M., and Terman, B. I. (1999). Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization. Oncogene 18, 1619-1627. Downward, J. (1998). Mechanisms and consequences of activation of protein kinase B/Akt. Curr Opin Cell Biol 10, 262-267. Du, R., Lu, K. V., Petritsch, C., Liu, P., Ganss, R., Passegue, E., Song, H., Vandenberg, S., Johnson, R. S., Werb, Z., and Bergers, G. (2008). HIFlalpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell 13, 206-220. Dumont, D. J., Gradwohl, G., Fong, G. H., Puri, M. C., Gertsenstein, M., Auerbach, A., and Breitman, M. L. (1994). Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev 8, 1897-1909. Duval, M., Bedard-Goulet, S., Delisle, C., and Gratton, J. P. (2003). Vascular endothelial growth factor-dependent down-regulation of Flk-l/KDR involves Cbl- 155 mediated ubiquitination. Consequences on nitric oxide production from endothelial cells. J Biol Chem 278, 20091-20097. Dvorak, H. F. (2002). Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol 20, 4368-4380. Eberhard, A., Kahlert, S., Goede, V., Hemmerlein, B., Plate, K. H., and Augustin, H. G. (2000). Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res 60, 1388-1393. Ebos, J. M„ Lee, C. R., and Kerbel, R. S. (2009). Tumor and host-mediated pathways of resistance and disease progression in response to antiangiogenic therapy. Clin Cancer Res 15, 5020-5025. Eklund, L., and Olsen, B. R. (2006). Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling. Exp Cell Res 312, 630- 641. Emmenegger, U., Man, S., Shaked, Y., Francia, G., Wong, J. W., Hicklin, D. J., and Kerbel, R. S. (2004). A comparative analysis of low-dose metronomic cyclophosphamide reveals absent or low-grade toxicity on tissues highly sensitive to the toxic effects of maximum tolerated dose regimens. Cancer Res 64, 3994- 4000. Erber, R., Thurnher, A., Katsen, A. D., Groth, G., Kerger, H., Hammes, H. P., Menger, M. D., Ullrich, A., and Vajkoczy, P. (2004). Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms. FASEB J 18, 338-340. Ettenberg, S. A., Rubinstein, Y. R., Banerjee, P., Nau, M. M., Keane, M. M., and Lipkowitz, S. (1999). cbl-b inhibits EGF-receptor-induced apoptosis by enhancing ubiquitination and degradation of activated receptors. Mol Cell Biol Res Commun 2, 111-118. Fan F, Wey JS, McCarty MF, Belcheva A, Liu W. (2005) Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells. Oncogene 24: 2647-2653. Fan, F., Stoeltzing, O., Liu, W., McCarty, M. F., Jung, Y. D., Reinmuth, N., and Ellis, L. M. (2004). Interleukin-lbeta regulates angiopoietin-1 expression in human endothelial cells. Cancer Res 64, 3186-3190. Fathers, K. E., Stone, C. M., Minhas, K., Marriott, J. J., Greenwood, J. D., Dumont, D. J., and Coomber, B. L. (2005). Heterogeneity of Tie2 expression in tumor microcirculation: influence of cancer type, implantation site, and response to therapy. Am J Pathol 167, 1753-1762. Feng Y, Venema VJ, Venema RC, Tsai N, Caldwell RB. (1999) VEGF induces nuclear translocation of Flk-l/KDR, endothelial nitric oxide synthase, and caveolin-1 in vascular endothelial cells. Biochem Biophys Res Commun; 256:192-7. Ferrara, N. (2000). Vascular endothelial growth factor and the regulation of angiogenesis. Recent Prog Horm Res 55, 15-35; discussion 35-16. Ferrara, N. (2002). VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer 2, 795-803. 156 Ferrara, N., Gerber, H. P., and LeCouter, J. (2003). The biology of VEGF and its receptors. Nat Med 9, 669-676. Ferrara, N., Hillan, K. J., and Novotny, W. (2005). Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 333, 328-335. Ferrer, F. A., Miller, L. J., Lindquist, R., Kowalczyk, P., Laudone, V. P., Albertsen, P. C., and Kreutzer, D. L. (1999). Expression of vascular endothelial growth factor receptors in human prostate cancer. Urology 54, 567-572. Fiedler, U., Scharpfenecker, M., Koidl, S., Hegen, A., Grunow, V., Schmidt, J. M., Kriz, W„ Thurston, G., and Augustin, H. G. (2004). The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies. Blood 103, 4150-4156. Findley, C. M., Cudmore, M. J., Ahmed, A., and Kontos, C. D. (2007). VEGF induces Tie2 shedding via a phosphoinositide 3-kinase/Akt dependent pathway to modulate Tie2 signaling. Arterioscler Thromb Vase Biol 27, 2619-2626. Flamme, I., and Risau, W. (1992). Induction of vasculogenesis and hematopoiesis in vitro. Development 116, 435-439. Folberg, R., Hendrix, M. J., and Maniotis, A. J. (2000). Vasculogenic mimicry and tumor angiogenesis. Am J Pathol 156, 361-381. Folberg, R., and Maniotis, A. J. (2004). Vasculogenic mimicry. APMIS 112, 508- 525. Folkman, J. (1971). Tumor angiogenesis: therapeutic implications. N Engl J Med 285, 1182-1186. Fong, G. H., Rossant, J., Gertsenstein, M., and Breitman, M. L. (1995). Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376, 66-70. Fontanini, G., Boldrini, L., Vignati, S., Chine, S., Basolo, F., Silvestri, V., Lucchi, M., Mussi, A., Angeletti, C. A., and Bevilacqua, G. (1998). Bcl2 and p53 regulate vascular endothelial growth factor (VEGF)-mediated angiogenesis in non-small cell lung carcinoma. Eur J Cancer 34, 718-723. Fontanini, G., Lucchi, M., Vignati, S., Mussi, A., Ciardiello, F., De Laurentiis, M., De Placido, S„ Basolo, F„ Angeletti, C. A., and Bevilacqua, G. (1997a). Angiogenesis as a prognostic indicator of survival in non-small-cell lung carcinoma: a prospective study. J Natl Cancer Inst 89, 881-886. Fontanini, G., Vignati, S., Boldrini, L., Chine, S., Silvestri, V., Lucchi, M., Mussi, A., Angeletti, C. A., and Bevilacqua, G. (1997b). Vascular endothelial growth factor is associated with neovascularization and influences progression of non- small cell lung carcinoma. Clin Cancer Res 3, 861-865. Ford, E. C., Herman, J., Yorke, E., and Wahl, R. L. (2009). 18F-FDG PET/CT for image-guided and intensity-modulated radiotherapy. J Nucl Med 50, 1655-1665. Foye, L. V., Jr., Chapman, C. G„ Willett, F. M„ and Adams, W. S. (1960). Cyclophosphamide. A preliminary study of a new alkylating agent. Cancer Chemother Rep 6, 39-40. 157 Fukuhara, S., Sako, K., Noda, K., Zhang, J., Minami, M., and Mochizuki, N. (2010). Angiopoietin-1/Tie2 receptor signaling in vascular quiescence and angiogenesis. Histol Histopathol 25, 387-396. Gale, N. W., Thurston, G„ Hackett, S. F., Renard, R., Wang, Q., McClain, J., Martin, C., Witte, C., Witte, M. H., Jackson, D., et al. (2002). Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev Cell 3, 411-423. Gao, A. G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996). Thrombospondin modulates alpha v beta 3 function through integrin- associated protein. J Cell Biol 135, 533-544. Garber, K. (2002). Could less be more? Low-dose chemotherapy goes on trial. J Natl Cancer Inst 94, 82-84. Garcia-Saenz, J. A., Martin, M., Calles, A., Bueno, C., Rodriguez, L., Bobokova, J., Custodio, A., Casado, A., and Diaz-Rubio, E. (2008). Bevacizumab in combination with metronomic chemotherapy in patients with anthracycline- and taxane-refractory breast cancer. J Chemother 20, 632-639. Gasparini, G. (2001). Metronomic scheduling: the future of chemotherapy? Lancet Oncol 2, 733-740. Gasparini, G., Longo, R., Toi, M., and Ferrara, N. (2005). Angiogenic inhibitors: a new therapeutic strategy in oncology. Nat Clin Pract Oncol 2, 562-577. Gatenby, R. A., and Gillies, R. J. (2004). Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4, 891-899. Gehling, U. M., Ergun, S., and Fiedler, W. (2007). CFU-EC: how they were originally defined. Blood 110, 1073. George, D. J., Halabi, S„ Shepard, T. F., Vogelzang, N. J., Hayes, D. F., Small, E. J., and Kantoff, P. W. (2001). Prognostic significance of plasma vascular endothelial growth factor levels in patients with hormone-refractory prostate cancer treated on Cancer and Leukemia Group B 9480. Clin Cancer Res 7, 1932- 1936. Gerber, D. E. (2008). Targeted therapies: a new generation of cancer treatments. Am Fam Physician 77, 311 -319. Gerber, H. P., and Ferrara, N. (2005). Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies. Cancer Res 65, 671-680. Gerber, H. P., Malik, A. K., Solar, G. P., Sherman, D., Liang, X. H., Meng, G„ Hong, K., Marsters, J. C., and Ferrara, N. (2002). VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism. Nature 417, 954-958. Gerber, H. P., McMurtrey, A., Kowalski, J., Yan, M., Keyt, B. A., Dixit, V., and Ferrara, N. (1998). Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway. Requirement for Flk-l/KDR activation. J Biol Chem 273, 30336-30343. Gerhardt, H., Golding, M., Fruttiger, M., Ruhrberg, C., Lundkvist, A., Abramsson, A., Jeltsch, M., Mitchell, C., Alitalo, K., Shima, D. and Betsholtz, C. (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J. Cell Biol. 161, 1163-1177 158 Gerhardt, H., and Betsholtz, C. (2005). How do endothelial cells orientate? EXS, 3-15. Giaccia, A. J., Brown, J. M., Wouters, B., Denko, N., and Koumenis, C. (1998). Cancer therapy and tumor physiology. Science 279, 12-13. Gitay-Goren, H., Halaban, R., and Neufeld, G. (1993). Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors. Biochem Biophys Res Commun 190, 702-708. Giuliani, N., Colla, S., Morandi, F., and Rizzoli, V. (2005). Angiopoietin-1 and myeloma-induced angiogenesis. Leuk Lymphoma 46, 29-33. Glade Bender, J., Cooney, E. M„ Kandel, J. J., and Yamashiro, D. J. (2004). Vascular remodeling and clinical resistance to antiangiogenic cancer therapy. Drug Resist Updat 7, 289-300. Gogvadze, V., Zhivotovsky, B., and Orrenius, S. (2009). The Warburg effect and mitochondrial stability in cancer cells. Mol Aspects Med. Gorski, D. H., Leal, A. D., and Goydos, J. S. (2003). Differential expression of vascular endothelial growth factor-A isoforms at different stages of melanoma progression. J Am Coll Surg 197, 408-418. Goundiam, O., Nagel, M. D., and Vayssade, M. (2009). Growth and survival signaling in B16F10 melanoma cells in 3D culture. Cell Biol Int. Graeber, T. G., Osmanian, C., Jacks, T., Housman, D. E., Koch, C. J., Lowe, S. W., and Giaccia, A. J. (1996). Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 379, 88-91. Graells, J., Vinyals, A., Figueras, A., Llorens, A., Moreno, A., Marcoval, J., Gonzalez, F. J., and Fabra, A. (2004). Overproduction of VEGF concomitantly expressed with its receptors promotes growth and survival of melanoma cells through MAPK and PI3K signaling. J Invest Dermatol 123, 1151-1161. Graeven, U., Fiedler, W., Karpinski, S., Ergun, S., Kilic, N., Rodeck, U., Schmiegel, W., and Hossfeld, D. K. (1999). Melanoma-associated expression of vascular endothelial growth factor and its receptors FLT-1 and KDR. J Cancer Res Clin Oncol 125, 621-629. Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977). Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36, 59-74. Grant, S. W., Kyshtoobayeva, A. S., Kurosaki, T., Jakowatz, J., and Fruehauf, J. P. (1998). Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and metastatic progression in melanoma. Cancer Detect Prev 22, 185-194. Gravallese, E. M., Pettit, A. R., Lee, R., Madore, R., Manning, C., Tsay, A., Gaspar, J., Goldring, M. B., Goldring, S. R., and Oettgen, P. (2003). Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha. Ann Rheum Dis 62, 100-107. Grbovic, O. M., Basso, A. D., Sawai, A., Ye, Q., Friedlander, P., Solit, D., and Rosen, N. (2006). V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc Natl Acad Sci U S A 103, 57-62. 159 Greenaway, J., Lawler, J., Moorehead, R., Bornstein, P., Lamarre, J., and Petrik, J. (2007). Thrombospondin-1 inhibits VEGF levels in the ovary directly by binding and internalization via the low density lipoprotein receptor-related protein-1 (LRP-1). J Cell Physiol 210, 807-818. Greenberg, J. I., Shields, D. J., Barillas, S. G., Acevedo, L. M., Murphy, E., Huang, J., Scheppke, L., Stockmann, C., Johnson, R. S., Angle, N., and Cheresh, D. A. (2008). A role for VEGF as a negative regulator of pericyte function and vessel maturation. Nature 456, 809-813. Greenwalt, D. E., Lipsky, R. H., Ockenhouse, C. F., Ikeda, H., Tandon, N. N., and Jamieson, G. A. (1992). Membrane glycoprotein CD36: a review of its roles in adherence, signal transduction, and transfusion medicine. Blood 80, 1105-1115. Grugel, S., Finkenzeller, G., Weindel, K., Barleon, B., and Marme, D. (1995). Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells. J Biol Chem 270, 25915-25919. Guillemin, G. J., and Brew, B. J. (2004). Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. J Leukoc Biol 75, 388-397. Guo, N. H„ Krutzsch, H. C., Negre, E., Vogel, T., Blake, D. A., and Roberts, D. D. (1992). Heparin- and sulfatide-binding peptides from the type I repeats of human thrombospondin promote melanoma cell adhesion. Proc Natl Acad Sci U S A 89, 3040-3044. Gutierrez, M. E., Kummar, S., and Giaccone, G. (2009). Next generation oncology drug development: opportunities and challenges. Nat Rev Clin Oncol 6, 259-265. Gutman, M., Singh, R. K., Xie, K., Bucana, C. D., and Fidler, I. J. (1995). Regulation of interleukin-8 expression in human melanoma cells by the organ environment. Cancer Res 55, 2470-2475. Hamano, Y., Sugimoto, H., Soubasakos, M. A., Kieran, M., Olsen, B. R., Lawler, J., Sudhakar, A., and Kalluri, R. (2004). Thrombospondin-1 associated with tumor microenvironment contributes to low-dose cyclophosphamide-mediated endothelial cell apoptosis and tumor growth suppression. Cancer Res 64, 1570- 1574. Hanahan, D., Bergers, G., and Bergsland, E. (2000). Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Invest 105, 1045-1047. Hanahan, D., and Folkman, J. (1996). Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57- 70. Hangai, M„ Moon, Y. S„ Kitaya, N., Chan, C. K., Wu, D. Y., Peters, K. G., Ryan, S. J., and Hinton, D. R. (2001). Systemically expressed soluble Tie2 inhibits intraocular neovascularization. Hum Gene Ther 12, 1311-1321. Hanks, S. K., Ryzhova, L., Shin, N. Y., and Brabek, J. (2003). Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci 8, d982-996. 160 Harfouche, R., and Hussain, S. N. (2006). Signaling and regulation of endothelial cell survival by angiopoietin-2. Am J Physiol Heart Circ Physiol 291, HI 635- 1645. Harris, A. L., Reusch, P., Barleon, B., Hang, C., Dobbs, N., and Marme, D. (2001). Soluble Tie2 and Fltl extracellular domains in serum of patients with renal cancer and response to antiangiogenic therapy. Clin Cancer Res 7, 1992- 1997. Hartmann, J. T., Haap, M., Kopp, H. G., and Lipp, H. P. (2009). Tyrosine kinase inhibitors - a review on pharmacology, metabolism and side effects. Curr Drug Metab 10, 470-481. Hashizume, H., Baluk, P., Morikawa, S., McLean, J. W., Thurston, G., Roberge, S., Jain, R. K., and McDonald, D. M. (2000). Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol 156, 1363-1380. Hata, K., Nakayama, K., Fujiwaki, R., Katabuchi, H., Okamura, H., and Miyazaki, K. (2004). Expression of the angopoietin-1, angopoietin-2, Tie2, and vascular endothelial growth factor gene in epithelial ovarian cancer. Gynecol Oncol 93, 215-222. Hayes, A. J., Huang, W. Q., Mallah, J., Yang, D., Lippman, M. E., and Li, L. Y. (1999). Angiopoietin-1 and its receptor Tie-2 participate in the regulation of capillary-like tubule formation and survival of endothelial cells. Microvasc Res 58, 224-237. Hellstrom, M., Kalen, M., Lindahl, P., Abramsson, A., and Betsholtz, C. (1999). Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126, 3047-3055. Hicklin, D. J., and Ellis, L. M. (2005). Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 23, 1011-1027. Hillen, F., and Griffioen, A. W. (2007). Tumour vascularization: sprouting angiogenesis and beyond. Cancer Metastasis Rev 26, 489-502. Hiratsuka, S., Mara, Y., Okada, A., Seiki, M., Noda, T., and Shibuya, M. (2001). Involvement of Fit-1 tyrosine kinase (vascular endothelial growth factor receptor- 1) in pathological angiogenesis. Cancer Res 61, 1207-1213. Hladovec, J. (1978). Circulating endothelial cells as a sign of vessel wall lesions. Physiol Bohemoslov 27, 140-144. Hlatky, L., Hahnfeldt, P., and Folkman, J. (2002). Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us. J Natl Cancer Inst 94, 883-893. Hobson, B., and Denekamp, J. (1984). Endothelial proliferation in tumours and normal tissues: continuous labelling studies. Br J Cancer 49, 405-413. Hockel, M., and Vaupel, P. (2001a). Biological consequences of tumor hypoxia. Semin Oncol 28, 36-41. Hockel, M., and Vaupel, P. (2001b). Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 93, 266-276. Hoffmann, J., Feng, Y., vom Hagen, F., Hillenbrand, A., Lin, J., Erber, R., Vajkoczy, P., Gourzoulidou, E., Waldmann, H., Giannis, A., et al. (2005). 161 Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity. FASEB J 19, 2035-2036. Holash, J., Wiegand, S. J., and Yancopoulos, G. D. (1999). New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 18, 5356-5362. Holopainen, T., Huang, H., Chen, C., Kim, K. E., Zhang, L., Zhou, F., Han, W., Li, C., Yu, J., Wu, J., et al. (2009). Angiopoietin-1 overexpression modulates vascular endothelium to facilitate tumor cell dissemination and metastasis establishment. Cancer Res 69, 4656-4664. Houck, K. A., Ferrara, N., Winer, J., Cachianes, G., Li, B., and Leung, D. W. (1991). The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5, 1806-1814. Hristov, M., and Weber, C. (2004). Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med 8, 498-508. Hsu, J. Y., and Wakelee, H. A. (2009). Monoclonal antibodies targeting vascular endothelial growth factor: current status and future challenges in cancer therapy. BioDrugs 23, 289-304. Hurwitz, H. (2004). Integrating the anti-VEGF-A humanized monoclonal antibody bevacizumab with chemotherapy in advanced colorectal cancer. Clin Colorectal Cancer 4 Suppl 2, S62-68. Hurwitz, H., Fehrenbacher, L., Novotny, W., Cartwright, T., Hainsworth, J., Heim, W., Berlin, J., Baron, A., Griffmg, S., Holmgren, E., et al. (2004). Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350, 2335-2342. Ikeda, N., Adachi, M., Taki, T., Huang, C, Hashida, H., Takabayashi, A., Sho, M., Nakajima, Y., Kanehiro, H., Hisanaga, M., et al. (1999). Prognostic significance of angiogenesis in human pancreatic cancer. Br J Cancer 79, 1553- 1563. Imanishi, Y., Hu, B., Jarzynka, M. J., Guo, P., Elishaev, E., Bar-Joseph, I., and Cheng, S. Y. (2007). Angiopoietin-2 stimulates breast cancer metastasis through the alpha(5)beta(l) integrin-mediated pathway. Cancer Res 67, 4254-4263. Ingram, D. A., Mead, L. E., Moore, D. B., Woodard, W., Fenoglio, A., and Yoder, M. C. (2005). Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells. Blood 105, 2783-2786. Ingram, D. A., Mead, L. E., Tanaka, H., Meade, V., Fenoglio, A., Mortell, K., Pollok, K., Ferkowicz, M. J., Gilley, D., and Yoder, M. C. (2004). Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104, 2752-2760. Inoue, S., Hartman, A., Branch, C. D., Bucana, C. D., Bekele, B. N., Stephens, L. C, Chada, S., and Ramesh, R. (2007). mda-7 In combination with bevacizumab treatment produces a synergistic and complete inhibitory effect on lung tumor xenograft. Mol Ther 15, 287-294. Ishizu, K., Sunose, N., Yamazaki, K., Tsuruo, T., Sadahiro, S., Makuuchi, H., and Yamori, T. (2007). Development and characterization of a model of liver 162 metastasis using human colon cancer HCT-116 cells. Biol Pharm Bull 30, 1779- 1783. • Ito, H., Rovira, II, Bloom, M. L., Takeda, K., Ferrans, V. J., Quyyumi, A. A., and Finkel, T. (1999). Endothelial progenitor cells as putative targets for angiostatin. Cancer Res 59, 5875-5877. • Iurlaro, M., Scatena, M., Zhu, W. H., Fogel, E., Wieting, S. L., and Nicosia, R. F. (2003). Rat aorta-derived mural precursor cells express the Tie2 receptor and respond directly to stimulation by angiopoietins. J Cell Sci 116, 3635-3643. • Iqbal J, Sun L, Zaidi M.Complexity in signal transduction. (2010) Ann N Y Acad Sci. 1192(l):238-44. • Ivy, S. P., Wick, J. Y., and Kaufman, B. M. (2009). An overview of small- molecule inhibitors of VEGFR signaling. Nat Rev Clin Oncol 6, 569-579. • Iwakura, A., Luedemann, C., Shastry, S., Hanley, A., Kearney, M., Aikawa, R., Isner, J. M., Asahara, T., and Losordo, D. W. (2003). Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury. Circulation 108, 3115-3121. • Jadvar, H., Alavi, A., and Gambhir, S. S. (2009). 18F-FDG uptake in lung, breast, and colon cancers: molecular biology correlates and disease characterization. J NuclMed 50, 1820-1827. • Jain, R. K. (2001). Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med 7, 987-989. • Jimenez, B., Volpert, O. V., Crawford, S. E., Febbraio, M., Silverstein, R. L., and Bouck, N. (2000). Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 6, 41-48. • Jones, N., and Dumont, D. J. (1998). The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R. Oncogene 17, 1097-1108. • Jones, N., and Dumont, D. J. (2000). Tek/Tie2 signaling: new and old partners. Cancer Metastasis Rev 19, 13-17. • Jung, Y. D., Ahmad, S. A., Akagi, Y., Takahashi, Y., Liu, W., Reinmuth, N., Shaheen, R. M., Fan, F., and Ellis, L. M. (2000). Role of the tumor microenvironment in mediating response to anti-angiogenic therapy. Cancer Metastasis Rev 19, 147-157. • Jung, Y. D., Ahmad, S. A., Liu, W., Reinmuth, N., Parikh, A., Stoeltzing, O., Fan, F., and Ellis, L. M. (2002). The role of the microenvironment and intercellular cross-talk in tumor angiogenesis. Semin Cancer Biol 12, 105-112. • Jurado, J. M., Sanchez, A., Pajares, B., Perez, E., Alonso, L., and Alba, E. (2008). Combined oral cyclophosphamide and bevacizumab in heavily pre-treated ovarian cancer. Clin Transl Oncol 10, 583-586. • Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V. W., Fang, G. H., Dumont, D., Breitman, M., and Alitalo, K. (1995). Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci U S A 92, 3566-3570. • Kalka, C., Masuda, H., Takahashi, T., Kalka-Moll, W. M., Silver, M., Kearney, M., Li, T., Isner, J. M., and Asahara, T. (2000a). Transplantation of ex vivo 163 expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A 97, 3422-3427. Kalka, C., Tehrani, H., Laudenberg, B., Vale, P. R., Isner, J. M., Asahara, T., and Symes, J. F. (2000b). VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease. Ann Thorac Surg 70, 829-834. Rallies, A., Rosenbauer, F., Scheller, M., Knobeloch, K. P., and Horak, I. (2002). Accumulation of c-Cbl and rapid termination of colony-stimulating factor 1 receptor signaling in interferon consensus sequence binding protein-deficient bone marrow-derived macrophages. Blood 99, 3213-3219. Kaneda, T., Sonoda, Y., Ando, K., Suzuki, T., Sasaki, Y., Oshio, T., Tago, M., and Kasahara, T. (2008). Mutation of Y925F in focal adhesion kinase (FAK) suppresses melanoma cell proliferation and metastasis. Cancer Lett 270, 354-361. Kaplan, R. N., Riba, R. D., Zacharoulis, S., Bramley, A. H., Vincent, L., Costa, C., MacDonald, D. D., Jin, D. K„ Shido, K., Kerns, S. A., et al. (2005). VEGFR 1- positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438, 820-827. Kasuga, M., Kahn, C. R., Hedo, J. A., Van Obberghen, E., and Yamada, K. M. (1981). Insulin-induced receptor loss in cultured human lymphocytes is due to accelerated receptor degradation. Proc Natl Acad Sci U S A 78, 6917-6921. Kawamoto, A., Gwon, H. C, Iwaguro, H., Yamaguchi, J. I., Uchida, S., Masuda, H„ Silver, M., Ma, H., Kearney, M„ Isner, J. M., and Asahara, T. (2001). Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation J 03, 634-637. Keating, M. T., and Williams, L. T. (1987). Processing of the platelet-derived growth factor receptor. Bio synthetic and degradation studies using anti-receptor antibodies. J Biol Chem 262, 7932-7937. Kerbel, R. S. (1991). Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents. Bioessays 13, 31-36. Kerbel, R. S., and Kamen, B. A. (2004). The anti-angiogenic basis of metronomic chemotherapy. Nat Rev Cancer 4, 423-436. Kerbel, R. S., Yu, J., Tran, J., Man, S., Viloria-Petit, A., Klement, G., Coomber, B. L., and Rak, J. (2001). Possible mechanisms of acquired resistance to anti- angiogenic drugs: implications for the use of combination therapy approaches. Cancer Metastasis Rev 20, 79-86. Kerkhoff, E., and Rapp, U. R. (1998). Cell cycle targets of Ras/Raf signalling. Oncogene 17, 1457-1462. Keyt, B. A., Berleau, L. T., Nguyen, H. V., Chen, H., Heinsohn, H., Vandlen, R., and Ferrara, N. (1996). The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem 271, 7788-7795. Kim, A., Balis, F. M., and Widemann, B. C. (2009). Sorafenib and sunitinib. Oncologist 14, 800-805. Kim, I., Kim, H. G„ So, J. N., Kim, J. H„ Kwak, H. J., and Koh, G. Y. (2000a). Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3'-Kinase/Akt signal transduction pathway. Circ Res 86, 24- 29. 164 Kim, I., Kim, J. H„ Ryu, Y. S., Jung, S. H„ Nah, J. J., and Koh, G. Y. (2000b). Characterization and expression of a novel alternatively spliced human angiopoietin-2. J Biol Chem 275, 18550-18556. Klement, G., Baruchel, S., Rak, J., Man, S., Clark, K., Hicklin, D. J., Bohlen, P., and Kerbel, R. S. (2000). Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 105, R15-24. Korpelainen, E. I., and Alitalo, K. (1998). Signaling angiogenesis and lymphangiogenesis. Curr Opin Cell Biol 10, 159-164. Krause, D. S., and Van Etten, R. A. (2005). Tyrosine kinases as targets for cancer therapy. N Engl J Med 353, 172-187. Kumar, I., Staton, C. A., Cross, S. S., Reed, M. W., and Brown, N. J. (2009). Angiogenesis, vascular endothelial growth factor and its receptors in human surgical wounds. Br J Surg 96, 1484-1491. Kunz, P., Hoffend, J., Altmann, A., Dimitrakopoulou-Strauss, A., Koczan, D., Eisenhut, M., Bonaterra, G. A., Dengler, T. J., Mier, W., Haberkorn, U., and Kinscherf, R. (2006). Angiopoietin-2 overexpression in morris hepatoma results in increased tumor perfusion and induction of critical angiogenesis-promoting genes. J Nucl Med 47, 1515-1524. Lacal, P. M., Ruffmi, F., Pagani, E., and D'Atri, S. (2005). An autocrine loop directed by the vascular endothelial growth factor promotes invasiveness of human melanoma cells. Int J Oncol 27, 1625-1632. Laderoute, K. R., Alarcon, R. M., Brody, M. D., Calaoagan, J. M., Chen, E. Y., Knapp, A. M., Yun, Z., Denko, N. C., and Giaccia, A. J. (2000). Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor. Clin Cancer Res 6, 2941- 2950. Lampugnani, M. G., Orsenigo, F., Gagliani, M. C., Tacchetti, C., and Dejana, E. (2006). Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J Cell Biol 174, 593-604. Langenberg, M. H., van Herpen, C. M., De Bono, J., Schellens, J. H., Unger, C., Hoekman, K., Blum, H. E., Fiedler, W., Drevs, J., Le Maulf, F„ et al. (2009). Effective strategies for management of hypertension after vascular endothelial growth factor signaling inhibition therapy: results from a phase II randomized, factorial, double-blind study of Cediranib in patients with advanced solid tumors. J Clin Oncol 27, 6152-6159. Larrivee, B., Niessen, K., Pollet, I., Corbel, S. Y., Long, M., Rossi, F. M., Olive, P. L., and Karsan, A. (2005). Minimal contribution of marrow-derived endothelial precursors to tumor vasculature. J Immunol 175, 2890-2899. Lartigau, E., Randrianarivelo, H., Avril, M. F., Margulis, A., Spatz, A., Eschwege, F., and Guichard, M. (1997). Intratumoral oxygen tension in metastatic melanoma. Melanoma Res 7, 400-406. Lawler, J. (2002). Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med 6, 1-12. 165 Lazar-Molnar, E., Hegyesi, H., Toth, S., and Falus, A. (2000). Autocrine and paracrine regulation by cytokines and growth factors in melanoma. Cytokine 12, 547-554. Le Tourneau, C., Raymond, E., and Faivre, S. (2007). Sunitinib: a novel tyrosine kinase inhibitor. A brief review of its therapeutic potential in the treatment of renal carcinoma and gastrointestinal stromal tumors (GIST). Ther Clin Risk Manag 3, 341-348. LeCouter, J., Moritz, D. R., Li, B., Phillips, G. L., Liang, X. H., Gerber, H. P., Hillan, K. J., and Ferrara, N. (2003). Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 299, 890-893. Lee HJ, Cho CH, Hwang SJ. (2004) Biological characterization ofangiopoietin-3 and angiopoietin-4. FASEB J 18:1200 - 8. Lee, B. A., Maher, D. W„ Hannink, M., and Donoghue, D. J. (1987). Identification of a signal for nuclear targeting in platelet-derived-growth-factor- related molecules. Mol Cell Biol 7, 3527-3537. Lee, T. H., Seng, S., Sekine, M., Hinton, C., Fu, Y., Avraham, H. K., and Avraham, S. (2007). Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR 1/FLT1. PLoS Med 4, el86. Lehman, T. A., Reddel, R., Peiifer, A. M., Spillare, E., Kaighn, M. E., Weston, A., Gerwin, B. I., and Harris, C. C. (1991). Oncogenes and tumor-suppressor genes. Environ Health Perspect 93, 133-144. Li, W., and Keller, G. (2000). VEGF nuclear accumulation correlates with phenotypical changes in endothelial cells. J Cell Sci 113 (Pt 9), 1525-1534. Liao, J. J., and Andrews, R. C. (2007). Targeting protein multiple conformations: a structure-based strategy for kinase drug design. Curr Top Med Chem 7, 1394- 1407. Liggett, W. H., Jr., and Sidransky, D. (1998). Role of the pl6 tumor suppressor gene in cancer. J Clin Oncol 16, 1197-1206. Lin, M. I., and Sessa, W. C. (2004). Antiangiogenic therapy: creating a unique "window" of opportunity. Cancer Cell 6, 529-531. Lin, P., Buxton, J. A., Acheson, A., Radziejewski, C., Maisonpierre, P. C., Yancopoulos, G. D., Channon, K. M., Hale, L. P., Dewhirst, M. W., George, S. E., and Peters, K. G. (1998a). Antiangiogenic gene therapy targeting the endothelium-specifk receptor tyrosine kinase Tie2. Proc Natl Acad Sci USA 95, 8829-8834. Lin, P., Polverini, P., Dewhirst, M., Shan, S., Rao, P. S., and Peters, K. (1997). Inhibition of tumor angiogenesis using a soluble receptor establishes a role for Tie2 in pathologic vascular growth. J Clin Invest 100, 2072-2078. Lin, P., Sankar, S., Shan, S., Dewhirst, M. W„ Polverini, P. J., Quinn, T. Q., and Peters, K. G. (1998b). Inhibition of tumor growth by targeting tumor endothelium using a soluble vascular endothelial growth factor receptor. Cell Growth Differ 9, 49-58. Lindahl, P., Hellstrom, M., Kalen, M., and Betsholtz, C. (1998). Endothelial- perivascular cell signaling in vascular development: lessons from knockout mice. Curr Opin Lipidol 9, 407-411. 166 Lindblom, P., Gerhardt, H., Liebner, S., Abramsson, A., Enge, M., Hellstrom, M., Backstrom, G., Fredriksson, S., Landegren, U., Nystrom, H. C., et al. (2003). Endothelial PDGF-B retention is required for proper investment of pericytes in the microvessel wall. Genes Dev 17, 1835-1840. Liu, B., Earl, H. M., Baban, D., Shoaibi, M., Fabra, A., Kerr, D. J., and Seymour, L. W. (1995). Melanoma cell lines express VEGF receptor KDR and respond to exogenously added VEGF. Biochem Biophys Res Commun 217, 721-727. Liu, W., Davis, D. W„ Ramirez, K., McConkey, D. J., and Ellis, L. M. (2001). Endothelial cell apoptosis is inhibited by a soluble factor secreted by human colon cancer cells. Int J Cancer 92, 26-30. Lobo, M., and Zachary, I. (2000). Nuclear localization and apoptotic regulation of an amino-terminal domain focal adhesion kinase fragment in endothelial cells. Biochem Biophys Res Commun 276, 1068-1074. Lobov, I. B., Brooks, P. C., and Lang, R. A. (2002). Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci U S A 99, 11205-11210. Loges, S., Mazzone, M., Hohensinner, P., and Carmeliet, P. (2009). Silencing or fueling metastasis with VEGF inhibitors: antiangiogenesis revisited. Cancer Cell 15, 167-170. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265-275. Lyden, D., Hattori, K., Dias, S., Costa, C., Blaikie, P., Butros, L., Chadburn, A., Heissig, B., Marks, W., Witte, L., et al. (2001). Impaired recruitment of bone- marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 7, 1194-1201. Macchiarini, P., Fontanini, G., Hardin, M. J., Squartini, F., and Angeletti, C. A. (1992). Relation of neovascularisation to metastasis of non-small-cell lung cancer. Lancet 340, 145-146. Machein, M. R., Knedla, A., Knoth, R., Wagner, S., Neuschl, E., and Plate, K. H. (2004). Angiopoietin-1 promotes tumor angiogenesis in a rat glioma model. Am J Pathol 165, 1557-1570. Maisonpierre, P. C., Suri, C., Jones, P. F., Bartunkova, S., Wiegand, S. J., Radziejewski, C, Compton, D., McClain, J., Aldrich, T. H., Papadopoulos, N., et al. (1997). Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277, 55-60. Makinde, T., and Agrawal, D. K. (2008). Intra and extravascular transmembrane signalling of angiopoietin-1-Tie2 receptor in health and disease. J Cell Mol Med 12, 810-828. Makinde, T., Murphy, R. F., and Agrawal, D. K. (2006). Immunomodulatory role of vascular endothelial growth factor and angiopoietin-1 in airway remodeling. CurrMol Med 6, 831-841. Man, S., Bocci, G., Francia, G., Green, S. K., Jothy, S., Hanahan, D., Bohlen, P., Hicklin, D. J., Bergers, G., and Kerbel, R. S. (2002). Antitumor effects in mice of low-dose (metronomic) cyclophosphamide administered continuously through the drinking water. Cancer Res 62, 2731-2735. 167 Mancuso, P., Calleri, A., Cassi, C., Gobbi, A., Capillo, M., Pruneri, G., Martinelli, G., and Bertolini, F. (2003). Circulating endothelial cells as a novel marker of angiogenesis. Adv Exp Med Biol 522, 83-97. Maniotis, A. J., Folberg, R., Hess, A., Seftor, E. A., Gardner, L. M., Pe'er, J., Trent, J. M., Meltzer, P. S., and Hendrix, M. J. (1999). Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol 155, 739-752. Martin-Pa dura, I., and Bertolini, F. (2009). Circulating endothelial cells as biomarkers for angiogenesis in tumor progression. Front Biosci (Schol Ed) 1, 304-318. Matsumoto, T., Bohman, S., Dixelius, J., Berge, T., Dimberg, A., Magnusson, P., Wang, L„ Wikner, C., Qi, J. H., Wernstedt, C., et al. (2005). VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis. EMBO J 24, 2342-2353. Matsumoto, T., and Mugishima, H. (2006). Signal transduction via vascular endothelial growth factor (VEGF) receptors and their roles in atherogenesis. J Atheroscler Thromb 13, 130-135. McCarthy, M. (2001). Targeted drugs take centre stage at US cancer meeting. Lancet 357, 1593. McCarthy, M. (2003). Antiangiogenesis drug promising for metastatic colorectal cancer. Lancet 361, 1959. Mendel, D. B., Laird, A. D., Xin, X., Louie, S. G., Christensen, J. G., Li, G., Schreck, R. E., Abrams, T. J., Ngai, T. J., Lee, L. B., et al. (2003). In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res 9, 327-337. Michallet, A. S., and Coiffier, B. (2009). Recent developments in the treatment of aggressive non-Hodgkin lymphoma. Blood Rev 23, 11-23. Michels, J., Lassau, N., Gross-Goupil, M., Massard, C., Mejean, A., and Escudier, B. (2009). Sunitinib inducing tumor lysis syndrome in a patient treated for renal carcinoma. Invest New Drugs. Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez, R., Moller, N. P., Risau, W., and Ullrich, A. (1993). High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72, 835-846. Miller, K., Wang, M„ Gralow, J., Dickler, M., Cobleigh, M., Perez, E. A., Shenkier, T., Cella, D., and Davidson, N. E. (2007). Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. N Engl J Med 357, 2666- 2676. Miyake, S„ Lupher, M. L., Jr., Druker, B., and Band, H. (1998). The tyrosine kinase regulator Cbl enhances the ubiquitination and degradation of the platelet- derived growth factor receptor alpha. Proc Natl Acad Sci U S A 95, 7927-7932. Miyanaga, N., and Akaza, H. (2009). [Sorafenib(Nexavar)]. Gan To Kagaku Ryoho 36, 1029-1033. 168 • Moon, W. S., Rhyu, K. H„ Kang, M. J., Lee, D. G., Yu, H. C„ Yeum, J. H., Koh, G. Y., and Tarnawski, A. S. (2003). Overexpression of VEGF and angiopoietin 2: a key to high vascularity of hepatocellular carcinoma? Mod Pathol 16, 552-557. • Moore, M. J. (1991). Clinical pharmacokinetics of cyclophosphamide. Clin Pharmacokinet 20, 194-208. • Morikawa, S., Baluk, P., Kaidoh, T., Haskell, A., Jain, R. K., and McDonald, D. M. (2002). Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors. Am J Pathol 160, 985-1000. • Motzer, R. J., Hoosen, S., Bello, C. L., and Christensen, J. G. (2006a). Sunitinib malate for the treatment of solid tumours: a review of current clinical data. Expert Opin Investig Drugs 15, 553-561. • Motzer, R. J., Michaelson, M. D., Redman, B. G., Hudes, G. R., Wilding, G„ Figlin, R. A., Ginsberg, M. S., Kim, S. T., Baum, C. M., DePrimo, S. E„ et al. (2006b). Activity of SU11248, a multitargeted inhibitor of vascular endothelial growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol 24, 16-24. • Mund, J. A., Ingram, D. A., Yoder, M. C., and Case, J. (2009). Endothelial progenitor cells and cardiovascular cell-based therapies. Cytotherapy 11, 103-113. • Munoz-Chapuli, R., Quesada, A. R., and Angel Medina, M. (2004). Angiogenesis and signal transduction in endothelial cells. Cell Mol Life Sci 61, 2224-2243. • Naiyer, A. J., Jo, D. Y., Ahn, J., Mohle, R., Peichev, M., Lam, G., Silverstein, R. L., Moore, M. A., and Rafii, S. (1999). Stromal derived factor-1-induced chemokinesis of cord blood CD34(+) cells (long-term culture-initiating cells) through endothelial cells is mediated by E-selectin. Blood 94, 4011-4019. • Natori, T., Sata, M., Washida, M., Hirata, Y., Nagai, R., and Makuuchi, M. (2002). G-CSF stimulates angiogenesis and promotes tumor growth: potential contribution of bone marrow-derived endothelial progenitor cells. Biochem Biophys Res Commun 297, 1058-1061. • Neagoe, P. E., Brkovic, A., Hajjar, F., and Sirois, M. G. (2009). Expression and release of angiopoietin-1 from human neutrophils: intracellular mechanisms. Growth Factors 27, 335-344. • Neuchrist, C., Erovic, B. M., Handisurya, A., Steiner, G. E., Rockwell, P., Gedlicka, C., and Burian, M. (2001). Vascular endothelial growth factor receptor 2 (VEGFR2) expression in squamous cell carcinomas of the head and neck. Laryngoscope 111, 1834-1841. • Neufeld, G., Cohen, T., Gengrinovitch, S., and Poltorak, Z. (1999). Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 13, 9-22. • Nilsson, I., Shibuya, M., and Wennstrom, S. (2004). Differential activation of vascular genes by hypoxia in primary endothelial cells. Exp Cell Res 299, 476- 485. • Nishishita, T„ and Lin, P. C. (2004). Angiopoietin 1, PDGF-B, and TGF-beta gene regulation in endothelial cell and smooth muscle cell interaction. J Cell Biochem 91, 584-593. • Nolan, D. J., Ciarrocchi, A., Mellick, A. S., Jaggi, J. S., Bambino, K., Gupta, S., Heikamp, E., McDevitt, M. R., Scheinberg, D. A., Benezra, R., and Mittal, V. 169 (2007). Bone marrow-derived endothelial progenitor cells are a major determinant of nascent tumor neovascularization. Genes Dev 21, 1546-1558. Nor, J. E., Mitra, R. S., Sutorik, M. M., Mooney, D. J., Castle, V. P., and Polverini, P. J. (2000). Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J Vase Res 37, 209-218. Oh, H., Takagi, H., Suzuma, K., Otani, A., Matsumura, M., and Honda, Y. (1999). Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells. J Biol Chem 274, 15732-15739. Olofsson, B., Korpelainen, E., Pepper, M. S., Mandriota, S. J., Aase, K., Kumar, V., Gunji, Y., Jeltsch, M. M., Shibuya, M., Alitalo, K, and Eriksson, U. (1998). Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells. Proc Natl Acad Sci U S A 95, 11709-11714. Orlic, D., Kajstura, J., Chimenti, S., Bodine, D. M., Leri, A., and Anversa, P. (2001). Transplanted adult bone marrow cells repair myocardial infarcts in mice. Ann N Y Acad Sci 938, 221-229; discussion 229-230. Paavonen, K., Puolakkainen, P., Jussila, L., Jahkola, T., and Alitalo, K. (2000). Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am J Pathol 156, 1499-1504. Paez-Ribes, M., Allen, E., Hudock, J., Takeda, T., Okuyama, H., Vinals, F., Inoue, M., Bergers, G., Hanahan, D., and Casanovas, O. (2009). Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15, 220-231. Papapetropoulos, A., Fulton, D., Mahboubi, K., Kalb, R. G., O'Connor, D. S., Li, F., Altieri, D. C., and Sessa, W. C. (2000). Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem 275, 9102-9105. Papapetropoulos, A., Garcia-Cardena, G., Dengler, T. J., Maisonpierre, P. C., Yancopoulos, G. D., and Sessa, W. C. (1999). Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors. Lab Invest 79, 213-223. Park, J. E„ Chen, H. H., Winer, J., Houck, K. A., and Ferrara, N. (1994). Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Fit-1 but not to Flk-l/KDR. J Biol Chem 269, 25646-25654. Park, J. E., Keller, G. A., and Ferrara, N. (1993). The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF. Mol Biol Cell 4, 1317-1326. Patenaude A, Parker J, Karsan A. (2010) Involvement of endothelial progenitor cells in tumor vascularization. Microvasc Res. 79(3):217-23. Pawson, T., and Scott, J. D. (1997). Signaling through scaffold, anchoring, and adaptor proteins. Science 278, 2075-2080. 170 Peters, K. G., Kontos, C. D., Lin, P. C., Wong, A. L., Rao, P., Huang, L., Dewhirst, M. W., and Sankar, S. (2004). Functional significance of Tie2 signaling in the adult vasculature. Recent Prog Horm Res 59, 51-71. Planque N. (2006) Nuclear trafficking of secreted factors and cell-surface receptors: new pathways to regulate cell proliferation and differentiation, and involvement in cancers. Cell Commun Signal.;4:7. Pollard, J. W. (2004). Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 4, 71-78. Poltorak, Z., Cohen, T., Sivan, R., Kandelis, Y., Spira, G., Vlodavsky, I., Keshet, E., and Neufeld, G. (1997). VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J Biol Chem 272, 7151-7158. Potgens, A. J., Lubsen, N. H., van Altena, M. C., Schoenmakers, J. G., Ruiter, D. J., and de Waal, R. M. (1995). Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice. Am J Pathol 146, 197-209. Punyadeera, C., Thijssen, V. L., Tchaikovski, S., Kamps, R., Delvoux, B., Dunselman, G. A., de Goeij, A. F„ Griffioen, A. W., and Groothuis, P. G. (2006). Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium. Mol Hum Reprod 12, 367-375. Purhonen, S., Palm, J., Rossi, D., Kaskenpaa, N., Rajantie, I., Yla-Herttuala, S., Alitalo, K., Weissman, I. L., and Salven, P. (2008). Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth. ProcNatl Acad Sci U S A 105, 6620-6625. Pytel, D., Sliwinski, T., Poplawski, T., Ferriola, D., and Majsterek, I. (2009). Tyrosine kinase blockers: new hope for successful cancer therapy. Anticancer Agents Med Chem 9, 66-76. Qian, X., and Tuszynski, G. P. (1996). Expression of thrombospondin-1 in cancer: a role in tumor progression. Proc Soc Exp Biol Med 212, 199-207. Quesada, A. R., Medina, M. A., and Alba, E. (2007). Playing only one instrument may be not enough: limitations and future of the antiangiogenic treatment of cancer. Bioessays29, 1159-1168. Rafii, S., and Lyden, D. (2003). Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 9, 702-712. Rak, J., Filmus, J., Finkenzeller, G., Grugel, S., Marme, D., and Kerbel, R. S. (1995). Oncogenes as inducers of tumor angiogenesis. Cancer Metastasis Rev 14, 263-277. Rak, J., Filmus, J., and Kerbel, R. S. (1996). Reciprocal paracrine interactions between tumour cells and endothelial cells: the 'angiogenesis progression' hypothesis. Eur J Cancer 32A, 2438-2450. Ramanujan, S., Koenig, G. C., Padera, T. P., Stoll, B. R., and Jain, R. K. (2000). Local imbalance of proangiogenic and antiangiogenic factors: a potential mechanism of focal necrosis and dormancy in tumors. Cancer Res 60, 1442- 1448.Ran, S., Huang, X., Downes, A., and Thorpe, P. E. (2003). Evaluation of novel antimouse VEGFR2 antibodies as potential antiangiogenic or vascular targeting agents for tumor therapy. Neoplasia 5, 297-307. 171 Ranpura, V., Pulipati, B., Chu, D., Zhu, X., and Wu, S. (2010). Increased Risk of High-Grade Hypertension With Bevacizumab in Cancer Patients: A Meta- Analysis. Am J Hypertens. Rapp, U. R., Cleveland, J. L., Storm, S. M., Beck, T. W., and Huleihel, M. (1986). Transformation by raf and myc oncogenes. Princess Takamatsu Symp 17, 55-74. Re RN, Cook JL.The basis of an intracrine pharmacology. J Clin Pharmacol. 2008 Mar;48(3):344-50. Real C, Caiado F, Dias S. (2008) Endothelial progenitors in vascular repair and angiogenesis: how many are needed and what to do? Cardiovasc Hematol Disord Drug Targets. 8(3): 185-93. Reardon, D. A., Desjardins, A., Vredenburgh, J. J., Gururangan, S., Sampson, J. H., Sathornsumetee, S., McLendon, R. E., Herndon, J. E., 2nd, Marcello, J. E., Norfleet, J., et al. (2009). Metronomic chemotherapy with daily, oral etoposide plus bevacizumab for recurrent malignant glioma: a phase II study. Br J Cancer 101, 1986-1994. Reed, E., Kohn, E. C., Sarosy, G., Dabholkar, M., Davis, P., Jacob, J., and Maher, M. (1995). Paclitaxel, cisplatin, and cyclophosphamide in human ovarian cancer: molecular rationale and early clinical results. Semin Oncol 22, 90-96. Ren, B., Yee, K. O., Lawler, J., and Khosravi-Far, R. (2006). Regulation of tumor angiogenesis by thrombospondin-1. Biochim Biophys Acta 1765, 178-188. Ribatti, D., Urbinati, C., Nico, B., Rusnati, M., Roncali, L., and Presta, M. (1995). Endogenous basic fibroblast growth factor is implicated in the vascularization of the chick embryo chorioallantoic membrane. Dev Biol 170, 39-49. Richter, M., and Zhang, H. (2005). Receptor-targeted cancer therapy. DNA Cell Biol 24, 271-282. Risau, W. (1997). Mechanisms of angiogenesis. Nature 386, 671-674. Risau, W., and Flamme, I. (1995). Vasculogenesis. Annu Rev Cell Dev Biol 11, 73-91. Robinson, R. S., Woad, K. J., Hammond, A. J., Laird, M., Hunter, M. G., and Mann, G. E. (2009). Angiogenesis and vascular function in the ovary. Reproduction 138, 869-881. Rodeck, U., Herlyn, M., Menssen, H. D., Furlanetto, R. W., and Koprowsk, H. (1987). Metastatic but not primary melanoma cell lines grow in vitro independently of exogenous growth factors. Int J Cancer 40, 687-690. Rodriguez-Manzaneque, J. C., Lane, T. F., Ortega, M. A., Hynes, R. O., Lawler, J., and Iruela-Arispe, M. L. (2001). Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor. Proc Natl Acad Sci U S A 98, 12485-12490. Rofstad, E. K., Galappathi, K., and Mathiesen, B. (2004). Thrombospondin-1 treatment prevents growth of dormant lung micrometastases after surgical resection and curative radiation therapy of the primary tumor in human melanoma xenografts. Int J Radiat Oncol Biol Phys 58, 493-499. 172 Ruiter, D. J., Schlingemann, R. O., Westphal, J. R., Denijn, M., Rietveld, F. J., and De Waal, R. M. (1993). Angiogenesis in wound healing and tumor metastasis. Beliring Inst Mitt, 258-272. Ruhrberg, C., Gerhardt, H., Golding, M., Watson, R., Ioannidou, S., Fujisawa, H., Betsholtz, C. and Shima, D.T. (2002) Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev. 16, 2684-2698 Salven, P., Heikkila, P., and Joensuu, H. (1997). Enhanced expression of vascular endothelial growth factor in metastatic melanoma. Br J Cancer 76, 930-934. Santos SC, Dias S. (2004). Internal and external autocrine VEGF/KDR loops regulate survival of subsets of acute leukemia through distinct signaling pathways. Blood 103:3883-3889 Sato, Y., and Rifkin, D. B. (1989). Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J Cell Biol 109, 309- 315. Sattler, U. G., Hirschhaeusera, F„ and Mueller-Klieser, W. F. (2009). Manipulation of Glycolysis in Malignant Tumors: Fantasy or Therapy? Curr Med Chem. Schlessinger, J. (2000). Cell signaling by receptor tyrosine kinases. Cell 103, 211- 225. Schmeisser, A., Garlichs, C. D., Zhang, H., Eskafi, S., Graffy, C., Ludwig, J., Strasser, R. H., and Daniel, W. G. (2001). Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions. Cardiovasc Res 49, 671-680. Schlessinger J. (2000) Cell signaling by receptor tyrosine kinases. Cell. 103(2):211-25. Schnurch, H., and Risau, W. (1993). Expression of tie-2, a member of a novel family of receptor tyrosine kinases, in the endothelial cell lineage. Development 119, 957-968. Schoffski, P., Bukowski, R., Flodgren, P., and Ravaud, A. (2009). Tyrosine kinase inhibition in renal cell carcinoma and gastrointestinal stromal tumours: case reports. Ann Oncol 20 Suppl 1, i25-i30. Scott, E. N., Meinhardt, G., Jacques, C., Laurent, D., and Thomas, A. L. (2007). Vatalanib: the clinical development of a tyrosine kinase inhibitor of angiogenesis in solid tumours. Expert Opin Investig Drugs 16, 367-379. Seftor, E. A., Meltzer, P. S., Schatteman, G. C., Gruman, L. M., Hess, A. R., Kirschmann, D. A., Seftor, R. E., and Hendrix, M. J. (2002). Expression of multiple molecular phenotypes by aggressive melanoma tumor cells: role in vasculogenic mimicry. Crit Rev Oncol Hematol 44, 17-27. Seftor, R. E., Seftor, E. A., Gehlsen, K. R., Stetler-Stevenson, W. G., Brown, P. D., Ruoslahti, E., and Hendrix, M. J. (1992). Role of the alpha v beta 3 integrin in human melanoma cell invasion. Proc Natl Acad Sci U S A 89, 1557-1561. Shahrara, S., Volin, M. V., Connors, M. A., Haines, G. K., and Koch, A. E. (2002). Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue. Arthritis Res 4, 201-208. 173 Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, X. F., Breitman, M. L., and Schuh, A. C. (1995). Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376, 62-66. Shvartsman SY, Hagan MP, Yacoub A, Dent P, Wiley HS, Lauffenburger DA. (2002)Autocrine loops with positive feedback enable context-dependent cell signaling. Am J Physiol Cell Physiol. 282(3):C545-59. Sher, I., Adham, S. A., Petrik, J., and Coomber, B. L. (2009). Autocrine VEGF- A/KDR loop protects epithelial ovarian carcinoma cells from anoikis. Int J Cancer 124, 553-561. Shi, Q., Rafii, S., Wu, M. H., Wijelath, E. S., Yu, C., Ishida, A., Fujita, Y., Kothari, S., Mohle, R., Sauvage, L. R., et al. (1998). Evidence for circulating bone marrow-derived endothelial cells. Blood 92, 362-367. Shibuya, M., Yamaguchi, S., Yamane, A., Ikeda, T., Tojo, A., Matsushime, H., and Sato, M. (1990). Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (fit) closely related to the fins family. Oncogene 5, 519-524. Shim, W. S., Teh, M., Bapna, A., Kim, I., Koh, G. Y., Mack, P. O., and Ge, R. (2002). Angiopoietin 1 promotes tumor angiogenesis and tumor vessel plasticity of human cervical cancer in mice. Exp Cell Res 279, 299-309. Shimoda, H., Bernas, M. J., Witte, M. H., Gale, N. W., Yancopoulos, G. D., and Kato, S. (2007). Abnormal recruitment of periendothelial cells to lymphatic capillaries in digestive organs of angiopoietin-2-deficient mice. Cell Tissue Res 328, 329-337. Shintani, S., Murohara, T., Ikeda, H., Ueno, T., Honma, T., Katoh, A., Sasaki, K., Shimada, T., Oike, Y., and Imaizumi, T. (2001). Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation 103, 2776-2779. Shord, S. S., Bressler, L. R., Tierney, L. A., Cuellar, S., and George, A. (2009). Understanding and managing the possible adverse effects associated with bevacizumab. Am J Health Syst Pharm 66, 999-1013. Shweiki, D., Neeman, M., Itin, A., and Keshet, E. (1995). Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis. Proc Natl Acad Sci U S A 92, 768-772. Siemeister, G., Schirner, M., Weindel, K., Reusch, P., Menrad, A., Marme, D., and Martiny-Baron, G. (1999). Two independent mechanisms essential for tumor angiogenesis: inhibition of human melanoma xenograft growth by interfering with either the vascular endothelial growth factor receptor pathway or the Tie-2 pathway. Cancer Res 5P, 3185-3191. Silverstein, R. L., and Febbraio, M. (2009). CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Sci Signal 2, re3. Simpkins, F., Belinson, J. L., and Rose, P. G. (2007). Avoiding bevacizumab related gastrointestinal toxicity for recurrent ovarian cancer by careful patient screening. Gynecol Oncol 107, 118-123. 174 Singh, H., Milner, C. S., Aguilar Hernandez, M. M., Patel, N., and Brindle, N. P. (2009). Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases. Cell Signal 21, 1346-1350. Slamon, D. J., Leyland-Jones, B., Shak, S., Fuchs, H., Paton, V., Bajamonde, A., Fleming, T., Eiermann, W., Wolter, J., Pegram, M., et al. (2001). Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344, 783-792. Smadja, D. M., Cornet, A., Emmerich, J., Aiach, M., and Gaussem, P. (2007). Endothelial progenitor cells: characterization, in vitro expansion, and prospects for autologous cell therapy. Cell Biol Toxicol 23, 223-239. Smalley, K. S., Contractor, R., Haass, N. K., Kulp, A. N., Atilla-Gokcumen, G. E., Williams, D. S., Bregman, H., Flaherty, K. T., Soengas, M. S., Meggers, E., and Herlyn, M. (2007). An organometallic protein kinase inhibitor pharmacologically activates p53 and induces apoptosis in human melanoma cells. Cancer Res 67, 209-217. Smith, J. K., Mamoon, N. M., and Duhe, R. J. (2004). Emerging roles of targeted small molecule protein-tyrosine kinase inhibitors in cancer therapy. Oncol Res 14, 175-225. Smolej, L. (2009). Modern concepts in the treatment of chronic lymphocytic leukemia. Hematology 14, 249-254. Solinas, G., Germano, G., Mantovani, A., and Allavena, P. (2009). Tumor- associated macrophages (TAM) as major players of the cancer-related inflammation. J Leukoc Biol 86, 1065-1073. Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., and Hebbel, R. P. (1997). Circulating activated endothelial cells in sickle cell anemia. N Engl J Med 337, 1584-1590. Sorkin, A., and Waters, C. M. (1993). Endocytosis of growth factor receptors. Bioessays 15, 375-382. Steeghs, N., Nortier, J. W., and Gelderblom, H. (2007). Small molecule tyrosine kinase inhibitors in the treatment of solid tumors: an update of recent developments. Ann Surg Oncol 14, 942-953. Stratmann, A., Acker, T., Burger, A. M., Amann, K., Risau, W., and Plate, K. H. (2001). Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants. Int J Cancer 91, 273-282. Straume, O., and Akslen, L. A. (2001). Expresson of vascular endothelial growth factor, its receptors (FLT-1, KDR) and TSP-1 related to microvessel density and patient outcome in vertical growth phase melanomas. Am J Pathol 159, 223-235. Suarez, H. G. (1989). Activated oncogenes in human tumors. Anticancer Res 9, 1331-1343. Sun, L., Liang, C, Shirazian, S., Zhou, Y., Miller, T., Cui, J., Fukuda, J. Y., Chu, J. Y., Nematalla, A., Wang, X., et al. (2003). Discovery of 5-[5-fluoro-2-oxo-l,2- dihydroindol-(3Z)-ylidenemethyl]-2,4- dimethyl-lH-pyrrole-3-carboxylic acid (2- diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase. J Med Chem 46, 1116-1119. 175 Sun, T„ Zhao, N., Ni, C. S., Zhao, X. L., Zhang, W. Z., Su, X., Zhang, D. F., Gu, Q., and Sun, B. C. (2009). Doxycycline inhibits the adhesion and migration of melanoma cells by inhibiting the expression and phosphorylation of focal adhesion kinase (FAK). Cancer Lett 285, 141-150. Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N., and Yancopoulos, G. D. (1996). Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171-1180. Takagi, H., Koyama, S., Seike, H., Oh, H., Otani, A., Matsumura, M., and Honda, Y. (2003). Potential role of the angiopoietin/tie2 system in ischemia-induced retinal neovascularization. Invest Ophthalmol Vis Sci 44, 393-402. Takahama, M., Tsutsumi, M., Tsujiuchi, T., Nezu, K., Kushibe, K., Taniguchi, S., Kotake, Y., and Konishi, Y. (1999). Enhanced expression of Tie2, its ligand angiopoietin-1, vascular endothelial growth factor, and CD31 in human non-small cell lung carcinomas. Clin Cancer Res 5, 2506-2510. Takahashi, M., Izawa, A., Ishigatsubo, Y., Fujimoto, K., Miyamoto, M., Horie, T., Aizawa, Y., Amano, J., Minota, S., Murohara, T., et al. (2009). Therapeutic neovascularization by the implantation of autologous mononuclear cells in patients with connective tissue diseases. Curr Pharm Des 15, 2778-2783. Takahashi, R., Tanaka, S., Kitadai, Y., Sumii, M., Yoshihara, M., Haruma, K., and Chayama, K. (2003). Expression of vascular endothelial growth factor and angiogenesis in gastrointestinal stromal tumor of the stomach. Oncology 64, 266- 274. Takahashi, T., Kalka, C., Masuda, H., Chen, D., Silver, M., Kearney, M., Magner, M., Isner, J. M., and Asahara, T. (1999). Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 5, 434-438. Takahashi, T., and Shibuya, M. (1997). The 230 kDa mature form of KDR/Flk-1 (VEGF receptor-2) activates the PLC-gamma pathway and partially induces mitotic signals in NIH3T3 fibroblasts. Oncogene 14, 2079-2089. Takahashi, T., Yamaguchi, S„ Chida, K., and Shibuya, M. (2001). A single autophosphorylation site on KDR/FIk-1 is essential for VEGF-A-dependent activation of PLC-gamma and DNA synthesis in vascular endothelial cells. EMBO J 20, 2768-2778. Takahashi, Y., Kitadai, Y„ Bucana, C. D., Cleary, K. R„ and Ellis, L. M. (1995). Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 55, 3964-3968. Takanami, I. (2004). Overexpression of Ang-2 mRNA in non-small cell lung cancer: association with angiogenesis and poor prognosis. Oncol Rep 12, 849- 853. Takimoto, C. H. (2009). Maximum tolerated dose: clinical endpoint for a bygone era? Target Oncol 4, 143-147. Tanaka, S., Mori, M., Sakamoto, Y., Makuuchi, M., Sugimachi, K., and Wands, J. R. (1999). Biologic significance of angiopoietin-2 expression in human hepatocellular carcinoma. J Clin Invest 103, 341-345. 176 Tanaka, S., Sugimachi, K., Yamashita Yi, Y., Ohga, T., Shirabe, K., Shimada, M., and Wands, J. R. (2002). Tie2 vascular endothelial receptor expression and function in hepatocellular carcinoma. Hepatology 35, 861-867. Taraboletti, G., Morbidelli, L., Donnini, S., Parenti, A., Granger, H. J., Giavazzi, R., and Ziche, M. (2000). The heparin binding 25 kDa fragment of thrombospondin-1 promotes angiogenesis and modulates gelatinase and TIMP-2 production in endothelial cells. FASEB J 14, 1674-1676. Tateishi-Yuyama, E., Matsubara, H., Murohara, T., Ikeda, U., Shintani, S., Masaki, H., Amano, K., Kishimoto, Y., Yoshimoto, K., Akashi, H„ et al. (2002). Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 360, 427-435. Teichert-Kuliszewska, K., Maisonpierre, P. C., Jones, N., Campbell, A. I., Master, Z., Bendeck, M. P., Alitalo, K., Dumont, D. J., Yancopoulos, G. D., and Stewart, D. J. (2001). Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2. Cardiovasc Res 49, 659-670. Thien, C. B., and Langdon, W. Y. (2001). Cbl: many adaptations to regulate protein tyrosine kinases. Nat Rev Mol Cell Biol 2, 294-307. Thigpen, J. T., Vance, R. B., and Khansur, T. (1993). Second-line chemotherapy for recurrent carcinoma of the ovary. Cancer 71, 1559-1564. Thurston, G., Suri, C„ Smith, K., McClain, J., Sato, T. N., Yancopoulos, G. D., and McDonald, D. M. (1999). Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286, 2511-2514. Tian, S., Hayes, A. J., Metheny-Barlow, L. J., and Li, L. Y. (2002). Stabilization of breast cancer xenograft tumour neovasculature by angiopoietin-1. Br J Cancer 86, 645-651. Timmermans, F., Plum, J., Yoder, M. C., Ingram, D. A., Vandekerckhove, B., and Case, J. (2009). Endothelial progenitor cells: identity defined? J Cell Mol Med 13, 87-102. Timmermans, F., Van Hauwermeiren, F., De Smedt, M., Raedt, R., Plasschaert, F., De Buyzere, M. L., Gillebert, T. C., Plum, J., and Vandekerckhove, B. (2007). Endothelial outgrowth cells are not derived from CD 133+ cells or CD45+ hematopoietic precursors. Arterioscler Thromb Vase Biol 27, 1572-1579. Torimura, T., Ueno, T., Kin, M., Harada, R., Taniguchi, E., Nakamura, T., Sakata, R., Hashimoto, O., Sakamoto, M., Kumashiro, R., et al. (2004). Overexpression of angiopoietin-1 and angiopoietin-2 in hepatocellular carcinoma. J Hepatol 40, 799-807. Trotter, M. J., Colwell, R., and Tron, V. A. (2003). Thrombospondin-1 and cutaneous melanoma. J Cutan Med Surg 7, 136-141. Tsai, J. C., Goldman, C. K., and Gillespie, G. Y. (1995). Vascular endothelial growth factor in human glioma cell lines: induced secretion by EGF, PDGF-BB, and bFGF. JNeurosurg 82, 864-873. Twardowski, P. W., Smith-Powell, L., Carroll, M., VanBalgooy, J., Ruel, C., Frankel, P., and Synold, T. W. (2008). Biologic markers of angiogenesis: circulating endothelial cells in patients with advanced malignancies treated on 177 phase I protocol with metronomic chemotherapy and celecoxib. Cancer Invest 26, 53-59. Vaklavas, C., Lenihan, D., Kurzrock, R., and Tsimberidou, A. M. (2010). Anti- vascular endothelial growth factor therapies and cardiovascular toxicity: what are the important clinical markers to target? Oncologist 15, 130-141. Valenzuela, D. M., Griffiths, J. A., Rojas, J., Aldrich, T. H., Jones, P. F., Zhou, H., McClain, J., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., et al (1999). Angiopoietins 3 and 4: diverging gene counterparts in mice and humans. Proc Natl Acad Sci U S A 96, 1904-1909. van der Geer, P., Hunter, T., and Lindberg, R. A. (1994). Receptor protein- tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10, 251-337. Vander Heiden, M. G., Cantley, L. C., and Thompson, C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324, 1029-1033. Varani, J., Riser, B. L., Hughes, L. A., Carey, T. E., Fligiel, S. E., and Dixit, V. M. (1989). Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells. Clin Exp Metastasis 7, 265-276. Vaupel, P., Hockel, M., and Mayer, A. (2007). Detection and characterization of tumor hypoxia using p02 histography. Antioxid Redox Signal 9, 1221-1235. Vaupel, P., Kallinowski, F., and Okunieff, P. (1989). Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 49, 6449-6465. Vaupel, P., Kelleher, D. K., and Hockel, M. (2001). Oxygen status of malignant tumors: pathogenesis of hypoxia and significance for tumor therapy. Semin Oncol 28, 29-35. Vaupel, P., and Mayer, A. (2005). Hypoxia and anemia: effects on tumor biology and treatment resistance. Transfus Clin Biol 12, 5-10. Venneri, M. A., De Palma, M., Ponzoni, M., Pucci, F., Scielzo, C., Zonari, E., Mazzieri, R., Doglioni, C., and Naldini, L. (2007). Identification of proangiogenic TIE2-expressing monocytes (TEMs) in human peripheral blood and cancer. Blood 109, 5276-5285. Vincent L, Jin DK, Karajannis MA, Shido K, Hooper AT. (2005) Fetal stromal- dependent paracrine and intracrine vascular endothelial growth factor-a/vascular endothelial growth factor receptor-1 signaling promotes proliferation and motility of human primary myeloma cells. Cancer Res 65: 3185-3192. Walter, D. H., and Dimmeler, S. (2002). Endothelial progenitor cells: regulation and contribution to adult neovascularization. Herz 27, 579-588. Wang, T. N., Qian, X., Granick, M. S., Solomon, M. P., Rothman, V. L., Berger, D. H., and Tuszynski, G. P. (1996). Thrombospondin-1 (TSP-1) promotes the invasive properties of human breast cancer. J Surg Res 63, 39-43. Warburg, O. (1961). [On the facultative anaerobiosis of cancer cells and its use in chemotherapy.]. Munch Med Wochenschr 103, 2504-2506. Warburg, O., Gawehn, K., Geissler, A. W., Kayser, D., and Lorenz, S. (1965). [EXPERIMENTS ON ANAEROBIOSIS OF CANCER CELLS.]. Klin Wochenschr 43, 289-293. 178 Waterhouse, C. (1974a). How tumors affect host metabolism. Ann N Y Acad Sci 230, 86-93. Waterhouse, C. (1974b). Lactate metabolism in patients with cancer. Cancer 33, 66-71. Weber, A., Pedrosa, I., Kawamoto, A., Himes, N., Munasinghe, J., Asahara, T., Rofsky, N. M., and Losordo, D. W. (2004). Magnetic resonance mapping of transplanted endothelial progenitor cells for therapeutic neovascularization in ischemic heart disease. Eur J Cardiothorac Surg 26, 137-143. Wedam, S. B., Low, J. A., Yang, S. X., Chow, C. K., Choyke, P., Danforth, D„ Hewitt, S. M., Berman, A., Steinberg, S. M., Liewehr, D. J., et al (2006). Antiangiogenic and antitumor effects of bevacizumab in patients with inflammatory and locally advanced breast cancer. J Clin Oncol 24, 769-777. Weidner, N., Carroll, P. R., Flax, J., Blumenfeld, W., and Folkman, J. (1993). Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol 143, 401-409. Whittles, C. E., Pocock, T. M., Wedge, S. R., Kendrew, J., Hennequin, L. F., Harper, S. J., and Bates, D. O. (2002). ZM323881, a novel inhibitor of vascular endothelial growth factor-receptor-2 tyrosine kinase activity. Microcirculation 9, 513-522. Wiley, H. S., and Burke, P. M. (2001). Regulation of receptor tyrosine kinase signaling by endocytic trafficking. Traffic 2, 12-18. Willett, C. G„ Boucher, Y., di Tomaso, E., Duda, D. G„ Munn, L. L., Tong, R. T., Chung, D. C., Sahani, D. V., Kalva, S. P., Kozin, S. V., et al (2004). Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 10, 145-147. Wilting, J., and Christ, B. (1996). Embryonic angiogenesis: a review. Naturwissenschaften 83, 153-164. Witzenbichler, B., Maisonpierre, P. C., Jones, P., Yancopoulos, G. D., and Isner, J. M. (1998). Chemotactic properties of angiopoietin-1 and -2, ligands for the endothelial-specific receptor tyrosine kinase Tie2. J Biol Chem 273, 18514- 18521. Wolfram, J. A., Diaconu, D., Hatala, D. A., Rastegar, J., Knutsen, D. A., Lowther, A., Askew, D., Gilliam, A. C., McCormick, T. S., and Ward, N. L. (2009). Keratinocyte but not endothelial cell-specific overexpression of Tie2 leads to the development of psoriasis. Am J Pathol 174, 1443-1458. Wong, A. L., Haroon, Z. A., Werner, S., Dewhirst, M. W., Greenberg, C. S., and Peters, K. G. (1997). Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues. Circ Res 81, 567-574. Xia, G., Kumar, S. R., Hawes, D., Cai, J., Hassanieh, L., Groshen, S., Zhu, S., Masood, R., Quinn, D. I., Broek, D., et al (2006). Expression and significance of vascular endothelial growth factor receptor 2 in bladder cancer. J Urol 175, 1245- 1252. Xu, Y., and Yu, Q. (2001). Angiopoietin-1, unlike angiopoietin-2, is incorporated into the extracellular matrix via its linker peptide region. J Biol Chem 276, 34990- 34998. 179 Yamakawa, M., Liu, L. X., Belanger, A. J., Date, T., Kuriyama, T., Goldberg, M. A., Cheng, S. H., Gregory, R. J., and Jiang, C. (2004). Expression of angiopoietins in renal epithelial and clear cell carcinoma cells: regulation by hypoxia and participation in angiogenesis. Am J Physiol Renal Physiol 287, F649-657. Yamazaki, Y., Tokunaga, Y., Takani, K., and Morita, T. (2005). Identification of the heparin-binding region of snake venom vascular endothelial growth factor (VEGF-F) and its blocking of VEGF-A165. Biochemistry 44, 8858-8864. Yana, I., Sagara, H., Takaki, S., Takatsu, K., Nakamura, K., Nakao, K., Katsuki, M., Taniguchi, S., Aoki, T., Sato, H., et al. (2007). Crosstalk between neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelial tip cells. J Cell Sci J20, 1607-1614. Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J., and Holash, J. (2000). Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248. Yang, J. C., Haworth, L., Sherry, R. M., Hwu, P., Schwartzentruber, D. J., Topalian, S. L., Steinberg, S. M., Chen, H. X., and Rosenberg, S. A. (2003). A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 349, 427-434. Yang, L., DeBusk, L. M., Fukuda, K., Fingleton, B., Green-Jarvis, B., Shyr, Y., Matrisian, L. M., Carbone, D. P., and Lin, P. C. (2004). Expansion of myeloid immune suppressor Gr+CDllb+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 6, 409-421. Yang, Z., Lei, Z., Li, B., Zhou, Y., Zhang, G. M., Feng, Z. H., Zhang, B., Shen, G. X., and Huang, B. (2009). Rapamycin inhibits lung metastasis of B16 melanoma cells through down-regulating alphav integrin expression and up- regulating apoptosis signaling. Cancer Sci. Yoder, M. C. (2009). Defining human endothelial progenitor cells. J Thromb Haemost 7 Suppl 1, 49-52. Yoder, M. C„ Mead, L. E., Prater, D., Krier, T. R, Mroueh, K. N., Li, F., Krasich, R., Temm, C. J., Prchal, J. T., and Ingram, D. A. (2007). Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals. Blood 109, 1801-1809. Yoshioka, T., Takahashi, M., Shiba, Y., Suzuki, C., Morimoto, H., Izawa, A., Ise, H., and Ikeda, U. (2006). Granulocyte colony-stimulating factor (G-CSF) accelerates reendothelialization and reduces neointimal formation after vascular injury in mice. Cardiovasc Res 70, 61-69. Yu, J. L., Rak, J. W., Carmeliet, P., Nagy, A., Kerbel, R. S„ and Coomber, B. L. (2001). Heterogeneous vascular dependence of tumor cell populations. Am J Pathol 158, 1325-1334. Yu, J. L., Rak, J. W., Coomber, B. L., Hicklin, D. J., and Kerbel, R. S. (2002). Effect of p53 status on tumor response to antiangiogenic therapy. Science 295, 1526-1528. Yu, Q., and Stamenkovic, I. (2001). Angiopoietin-2 is implicated in the regulation of tumor angiogenesis. Am J Pathol 158, 563-570. 180 Yuan, H. T., Khankin, E. V., Karumanchi, S. A., and Parikh, S. M. (2009). Angiopoietin 2 is a partial agonist/antagonist of Tie2 signaling in the endothelium. Mol Cell Biol 29, 2011-2022. Zabrenetzky, V., Harris, C. C., Steeg, P. S., and Roberts, D. D. (1994). Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung and breast carcinoma cell lines. Int J Cancer 59, 191-195. Zachary, I., and Gliki, G. (2001). Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res 49, 568-581. Zaghloul, N., Hernandez, S. L., Bae, J. O., Huang, J., Fisher, J. C., Lee, A., Kadenhe-Chiweshe, A., Kandel, J. J., and Yamashiro, D. J. (2009). Vascular endothelial growth factor blockade rapidly elicits alternative proangiogenic pathways in neuroblastoma. Int J Oncol 34, 401-407. Zangari, M., Fink, L. M., Elice, F., Zhan, F., Adcock, D. M., and Tricot, G. J. (2009). Thrombotic events in patients with cancer receiving antiangiogenesis agents. J Clin Oncol 27, 4865-4873. Zeng, H., Dvorak, H. F., and Mukhopadhyay, D. (2001a). Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) peceptor-1 down- modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways. J Biol Chem 276, 26969-26979. Zeng, H., Sanyal, S., and Mukhopadhyay, D. (2001b). Tyrosine residues 951 and 1059 of vascular endothelial growth factor receptor-2 (KDR) are essential for vascular permeability factor/vascular endothelial growth factor-induced endothelium migration and proliferation, respectively. J Biol Chem 276, 32714- 32719. Zhang, H., Li, Y., Li, H., Bassi, R., Jimenez, X., Witte, L., Bohlen, P., Hicklin, D., and Zhu, Z. (2004). Inhibition of both the autocrine and the paracrine growth of human leukemia with a folly human antibody directed against vascular endothelial growth factor receptor 2. Leuk Lymphoma 45, 1887-1897. Zhang, L., Yang, N., Park, J. W., Katsaros, D., Fracchioli, S., Cao, G., O'Brien- Jenkins, A., Randall, T. C, Rubin, S. C., and Coukos, G. (2003). Tumor-derived vascular endothelial growth factor up-regulates angiopoietin-2 in host endothelium and destabilizes host vasculature, supporting angiogenesis in ovarian cancer. Cancer Res 63, 3403-3412. Zhang, Z. L., Liu, Z. S., and Sun, Q. (2006). Expression of angiopoietins, Tie2 and vascular endothelial growth factor in angiogenesis and progression of hepatocellular carcinoma. World J Gastroenterol 12, 4241-4245. Zou, X., Lin, Y., Rudchenko, S., and Calame, K. (1997). Positive and negative regulation of c-Myc transcription. Curr Top Microbiol Immunol 224, 57-66. 181 APPENDIX I - Chemical List and Suppliers Acetone, 100% Fisher Scientific, Nepean, Ontario, Canada Acylamide/Bis Solution, 40% Biorad, Mississauga, ON, Canada Agarose Low EEO Electrophoresis Grade Fisher Scientific, Nepean, Ontario, Canada Amicon ultra centrifugal filters Milipore, Temecula, CA, USA Ammonium persulfate Sigma-Aldrich, Oakville, ON, Canada Antibody labeling kit, mouse, Alexa488 Invitrogen, Burlington, ON, Canada Aprotinin Sigma-Aldrich, Oakville, ON, Canada Aquapolymount Polyscience, Warrington, PA, USA Bovine Serum Albumin (BSA) Jackson ImmunoResearch Laboratories, West gRove, PA, USA Cell Culture Dishes (60 and 100mm) Sarstedt, Newton, NC, USA Cell lysis buffer, lOx Cell Signaling Technology, Danvers, MA, USA CellTracker Red CMPTX (577/602 nm) Invitrogen, Burlington, ON, Canada Kit Chemilluminescent substrate Roche, Laval, Quebec, Canada Chloroform Fisher Scientific, Nepean, Ontario, Canada Cloning Disks Sigma-Aldrich, Oakville, ON, Canada Collagen, type IV rat tail BD Biosciences, Mississauga, ON, Canada Coverslips Fisher Scientific, Nepean, Ontario, Canada Cryomatrix Fisher Scientific, Nepean, Ontario, Canada Dako pen Dako, Carpinteria, CA, USA Dako protein free block Dako, Carpinteria, CA, USA DAPI (4',6-diamidino-2-phenylindole Dako, Carpinteria, CA, USA Dc protein assay Biorad, Mississauga, ON, Canada Diaminobenzidine (DAB) Dimethyl Sulfoxide (DMSO) Fisher Scientific, Nepean, Ontario, Canada 100 bp DNA Ladder - Generuler MBI Fermentas, Burlington, ON, Canada dNTP lOOmM set Amersham, Biscataway, NJ, Canada Donkey anti-rat FITC conjugated antibody Jackson ImmunoResearch Laboratories, West gRove, PA, USA Dulbecco's modified Eagle's Medium Sigma-Aldrich, Oakville, ON, Canada (DMEM) Endothelial Growth Media (EGM-2MV) Lonza, Basel, Switzerland (Clonetics) Ethidium Bromide lOmg/ml Biorad, Mississauga, ON, Canada Fetal bovine serum (FBS) Invitrogen, Burlington, ON, Canada Fibronectin Sigm Fluorescent mounting media Dako, Carpinteria, CA, USA Gentamicin Sigma-Aldrich, Oakville, ON, Canada Glucose free-media Invitrogen, Burlington, ON, Canada Glycine Fisher Scientific, Nepean, Ontario, Canada 182 Goat anti-CD31 Santa Cruz, Santa Cruz, CA Goat anti-mouse Cy3 conjugated antibody Jackson ImmunoResearch Laboratories, West gRove, PA, USA Goat anti-rabbit Cy3 conjugated antibody Jackson ImmunoResearch Laboratories, West gRove, PA, USA Goat anti-mouse POD antibody Sigma-Aldrich, Oakville, ON, Canada Goat anti-rabbit POD antibody Sigma-Aldrich, Oakville, ON, Canada Goat anti-rat FITC antibody Jackson ImmunoResearch Laboratories, West gRove, PA, USA Hank's Buffered Salt Solution (HBSS) Invitrogen, Burlington, ON, Canada (Gibco) Hematoxylin Solution, Mayer's Sigma-Aldrich, Oakville, ON, Canada Hydrogen peroxide, 30% Fisher Scientific, Nepean, Ontario, Canada 4-hydroxycyclophosphamide (4-HC) NIOMECH, IIT GmbH, Bielefeld, Germany Hypoxyprobe-1 Chemicon International Inc, Temecula, CA, USA Isopropanol Fisher Scientific, Nepean, Ontario, Canada Matrigel BD Biosciences, Mississauga, ON, Canada Minimal Essential Medium (MEM), 1 OX Invitrogen, Burlington, ON, Canada Methanol, 100% Fisher Scientific, Nepean, Ontario, Canada Modular Incubator Chamber Billups-Rothenberg Inc, Del Mar, CA, USA Mouse anti-a-tubulin antibody Sigma-Aldrich, Oakville, ON, Canada Mouse anti-a-smooth muscle actin Sigma-Aldrich, Oakville, ON, Canada Mouse anti-goat POD Sigma-Aldrich, Oakville, ON, Canada Mouse anti-N-Cadherin BD Biosciences, Mississauga, ON, Canada Mouse anti-thrombospondin-1 Ab-2 (D4.6) Neomarkers, Fremont, CA Mouse anti-Tie2 antibody BD Biosciences, Mississauga, ON, Canada Mouse anti-Tie2 (AB33) Cell Signaling Technology, Danvers, MA, USA Normal goat serum Sigma-Aldrich, Oakville, ON, Canada Paraformaldehyde, 4% USB Corporation, Cleveland, OH, USA PMSF, powder Sigma-Aldrich, Oakville, ON, Canada PVDF membrane Roche, Laval, Quebec, Canada Rabbit anti-desmin antibody Abeam, Cambridge, MA, USA Rabbit anti-pan-Akt antibody Cell Signaling Technology, Danvers, MA, USA Rabbit anti-PDGFRp antibody Cell Signaling Technology, Danvers, MA, USA Rabbit anti-phospho-Akt (Ser473) antibody Cell Signaling Technology, Danvers, MA, USA Rabbit anti-phospho-Tie2 (Tyr992) Cell Signaling Technology, Danvers, MA, antibody USA 183 Rabbit anti-VEGFR2 Cell Signaling Technology, Danvers, MA, USA Rat anti-CD31 antibody Hycult Biotechnology, Uden, NL R.T.U Vectastain Elite ABC Reagent Vector, Burlington, Ontario, Canada SDS, 20% Biorad, Mississauga, ON, Canada Sodium Bicarbonate Solution Invitrogen, Burlington, ON, Canada Sodium Chloride Fisher Scientific, Nepean, Ontario, Canada Sodium Dedecyl Sulfate (SDS), 20% Biorad, Mississauga, ON, Canada Sodium Orthovanadate Fisher Scientific, Nepean, Ontario, Canada Sodium Pyruvate Invitrogen, Burlington, ON, Canada Streptavidin conjugated to Alexa350 Invitrogen, Burlington, ON, Canada Stripping Solution, Reblot Plus Milipore, Temecula, CA, USA Taq polymerase Invitrogen, Burlington, ON, Canada TekdeltaFc, murine Amgen, Thousand Oaks, CA, USA TEMED (N,N,N,N-Tetramethylethylene Roche, Laval, Quebec, Canada diamine) 0.5 M Tris-HCl Buffer pH 6.8 Biorad, Mississauga, ON, Canada 1.5 M Tris-HCl Buffer pH 8.8 Biorad, Mississauga, ON, Canada Tris, powder Fisher Scientific, Nepean, Ontario, Canada Triton X-100 non-ionic detergent Biorad, Mississauga, ON, Canada Trypan blue solution, 4% Sigma-Aldrich, Oakville, ON, Canada Turbo DNA-free Applied Biosciences/Ambion, Austin, TX Tween-20 Biorad, Mississauga, ON, Canada Vacutainer BD Biosciences, Mississauga, ON, Canada X-ray film Konica 184 APPENDIX II - Preparation of Materials Used 4M Ammonium Chloride Solution 154 mM ammonium chloride 10 mM potassium bicarbonate 0.1 mMEDTA Acid Citrate Dextrose (ACD) In 200 ml of water we dissolved 1.825 g citric acid (anhydrous) 5.500 g sodium citrate (dihydrous) 5.600 g dextrose Volume was adjusted to 250 ml, filter sterilized and stored at -20C. 2% agarose gel 2g of agarose and 100 ml of IX TBE buffer was added to the flask. Solution was heated in the microwave until completely dissolved and then allowed to slightly cool. Once cooled, 4 fil of ethidium bromide was added to the liquid and poured into casting apparatus. Gel was allowed to solidify before usage. Avertin To the beaker, 2.5g of 2,2,2-tribromoethanol, 5ml of 2-methyl-2-butanol was added and dissolved in the fumehood to a final solution of 200 ml. Solution was filter sterilized and stored in the dark at 4°C. Casting acrylamide gels for western blotting (1.5 mm thick) To make 7.5% acrylamide resolving (lower) gel the following amounts were used: Distilled water 11 ml 40% acrylamide solution 3.72 ml 1.5 MpH 8.8 Tris buffer 5 ml 10% SDS 200 \x\ TEMED 10 nl 10% Ammonium Persulfate 240 Once lower, resolving gel is firm, stacking gel was prepared and the following amounts used: Distilled water 3.2 ml 40% acrylamide solution 0.5 ml 1.5 M pH 6.8 Tris buffer 1.26 ml 185 10% SDS 50 nl TEMED 5 fal 10% Ammonium Persulfate 50 nl Plastic combs (1.5mm) were added to the gels and allowed to solidify. Gels were stored in wet tissue paper in plastic container up to a week in 4°C until use. Cell Lysis Buffer (Cell Signaling Technology) contents: 20 mM Tris-HCl (pH 7.5) 150 mM NaCl 1 mM Na2EDTA 1 mM EGTA 1% Triton-X 2.5 mM sodium pyrophosphate 1 mM beta-glycerophosphate 1 mM Na3V04 1 (ig/ml leupeptin 1 mM PMSF 2 ng/ml aprotinine Direct labeling of Hypoxyprobe-1 Antibody First, 2.0 of hypoxyprobe antibody was mixed in 17.5 |il of PBS and 5.0 jal of Component A (containing the green probe) and incubated in the dark at room temperature for 5 minutes. Next, 5.0 JLII of Component B (containing blocking solution to stop the reaction) was added and mixture was incubated for another 5 minutes. Volume was adjusted to 400.0 pi (final dilution of hypoxyprobe antibody is 1:200) and one drop of Dako protein-free blocking solution (Dako Cytomotation) was added. EGM-2MV (endothelial specific growth media) was supplemented with 2% fetal bovine serum, VEGF, IGF, FGF, EGF, hydrocortisone, antibiotics and ascorbic acid; Lonza, Basel, Switzerland). TBE buffer 10X To 400 ml of deionized water, 54 g of Tris base, 27.5g of boric acid and 20 ml of 0.5M EDTA (pH 8.0) was added. Solution was mixed to dissolve solids and final volume adjusted to 500 ml. Towbin Solution This solution was made by combining the following ingredients in 800 ml of distilled water: 30.250 g Tris-base 144.100 g Glycine 186 Final volume was adjusted to 1000 mL with distilled water and stored at 4°C. Transfer buffer 10X (for wet transfer of proteins) The following amounts were combined: 150 mL methanol 80 mL of Towbin solution 2 ml of 2% SDS Volume was adjusted to 1 liter with distilled water and chilled at 4°C for few hours before use. Tris Buffered Saline 10X (TBS) To make up this solution, the following ingredients were added to 800 ml of distilled water: 24.2 g Tris base 80 g NaCl First, pH was adjusted to 7.6 and then final volume was added to equal 1 liter. Wash Buffer TBS IX TBS buffer was prepared by combining 450 ml of distilled water and 50 ml of TBS (10X). Wash Buffer TBS/Tween lx TBS/Tween was prepared by combining 450 ml of miliQ water + 50 ml of TBS (lOx) + 500 pi of Tween-20 Blocking buffer(s) For blocking, 5% milk was prepared by mixing 100 ml of TBS/T and 5 g non-fat milk powder. 5% BSA blocking solution was prepared by mixing 100 ml of TBS/T and 5 g BSA powder. Loading buffer 5X for western blotting To prepare loading buffer for protein lysates the following solutions were added: 6.25 ml Tris buffer, pH 6.8 (1M) 0.5 ml EDTA (0.5M) pH 8 6.25 ml 20% SDS 10 ml Glycerol 1.5 ml 2% Bromopheno 1 Blue Final working solution was prepared by mixing 1 ml of loading buffer 5X and 50 JJ.1 P- mercaptoethanol. Preparing stripping solution and stripping the membranes of antibodies Antibody probed PVDF membranes were washed in TBS/Tween for 5 minutes. Striping Solution was prepared by diluting the stock solution 1:10 in distilled water. Membranes were incubated for 15 minutes in this stripping solution and then washed in 187 TBS/Tween. Next, membranes were blocked using 5% milk solution in TBS/T and protocol for western blotting was further followed as previously stated. Preparation of low dose Cyclophosphamide treatments for mouse trial Calculations were based on the assumption that mice drinks on average 1.5 ml/lOg/day. Since one mouse is about 25g, one mouse drinks about 3.75 ml/day. For metronomic cyclophosphamide treatments we used 30 mg of CTX/kg/day. According to the average weight of a mouse, one mouse needs 0.75 mg/day of cyclophosphamide which is 0.75 mg of CTX per 3.75 ml of drinking water. Therefore, the final calculation is that each mouse needs 0.2 mg/ml of CTX. Water bottles for mice hold about 200 ml so each bottle needed about 40 mg of CTX. Stock solution of 10 mg/ml was prepared and 4 ml of this solution was added to the drinking water of mice. Preparation of Tek deltaFc treatments for mouse trial Stock solution of murine TekdeltaFc ant ibody is 4.75 mg/ml. We injected 50 (al of TekdeltaFc per mouse which is about 250 |ig of TekdeltaFc per mouse. 188